Method for Measuring Glycoprotein, Method for Examining Liver Desease, Reagent for Quantitative Determination of Glycoprotein and Glycan-Marker Glycoprotein as an Index for Clinical Conditions of Liver Disease

ABSTRACT

An object of the present invention is to provide a method for measuring a glycan-marker glycoprotein, by which liver disease can be detected with higher accuracy than is possible with conventional methods. Also, an object of the present invention is to provide a method for examining liver disease, by which liver disease can be detected with higher accuracy than is possible with conventional methods. Also, an object of the present invention is to provide a reagent for quantitative determination of a glycoprotein, which is used for the above measurement methods. Furthermore, an object of the present invention is to provide a glycan-marker glycoprotein as an index for clinical conditions of liver disease, which is capable of identifying the clinical conditions of liver disease depending on the progress of liver disease. The method for measuring a glycoprotein is characterized in that: the glycoprotein is at least one glycoprotein selected from alpha-1-acid glycoprotein (AGP) and Mac-2-binding protein (M2BP) contained in a sample collected from a subject; when the glycoprotein is AGP, AGP binding to a first lectin selected from AOL and MAL is measured; and when the glycoprotein is M2BP, M2BP binding to a second lectin selected from WFA, BPL, AAL, RCA120, and TJAII is measured.

RELATED APPLICATIONS

This application is a continuation of PCT/JP2010/061891 filed on Jul.14, 2010, which claims priority to Japanese Application Nos. 2009-165795filed on Jul. 14, 2009 and 2009-287243 filed on Dec. 18, 2009. Theentire contents of these applications are incorporated herein byreference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for measuring at least oneglycoprotein selected from alpha-1-acid glycoprotein (AGP) andMac-2-binding protein (M2BP), a method for examining liver disease usingat least one glycoprotein selected from AGP and M2BP, and aglycan-marker glycoprotein with which hepatic cell carcinoma andbackground liver status such as regarding inflammation-fiber formationcan be detected based on glyco-alterations.

2. Description of the Related Art

Liver cancer can be roughly classified into primary liver cancer andmetastatic liver cancer developed in the liver. It is said that hepaticcell carcinoma accounts for 90% of primary liver cancer cases.

Hepatic cell carcinoma patients are often infected with hepatitis type Cvirus or hepatitis type B virus that constitutes the underlying cause ofa disease. The disease proceeds from acute viral hepatitis to chronicviral hepatitis, and then to cirrhosis. Long after the development ofviral hepatitis, the disease can become cancerous for the first time. Inthe case of cirrhosis, normal hepatocytes decrease through repetition ofinflammation and regeneration, and then the affected liver becomes anorgan composed of fibrous tissue. For example, there are said to be 3million type C hepatitis patients in Japan alone and 10 million or morein China and Africa. Also, in the case of type B and/or type C hepatitispatients, the carcinogenic rate from chronic hepatitis is an annual rateof 0.8% for mild chronic hepatitis (F1), an annual rate of 0.9% formoderate chronic hepatitis (F2), and an annual rate of 3.5% for severechronic hepatitis (F3). Furthermore, the probability of cirrhosis (F4)becoming cancerous increases to an annual rate of as high as 7% (FIG. 1and FIG. 2). Also, in the course of liver disease, firstly, functionsstart to disappear because of chronic hepatitis as the clinicalconditions proceed, pathological structure(s) appear because ofcirrhosis, and fiber formation of the liver proceeds. In this manner,the tissue image is altered (FIG. 3).

Early detection of cancer is important for cancer treatment. Also, inthe case of hepatic cell carcinoma, early detection of cancersignificantly influences treatment and postoperative prognosis. The5-year survival rate after hepatic resection is 80% for stage I cancerand only 38% for stage IV cancer.

As liver cancer markers, α-fetoprotein (AFP) and a protein induced byVitamin K absence or antagonist-II (PIVKA-II) are currently known(patent documents 1 and 2), but both the specificity and the sensitivitythereof are insufficient. Hence, physical examination for earlydetection of liver cancer is currently conducted with the use of livercancer markers and imaging studies such as ultrasonography, computedtomography (CT), and magnetic resonance imaging (MRI).

Also, non-patent document 1 describes an attempt to measure fucosylatedAGP in serum using AAL lectin so as to detect cirrhosis. However, thetechnique described in non-patent document 1 is unsatisfactory in viewof liver disease detection performance, since the specificity is about87% and the accuracy is about 77%, although the sensitivity is about66%, as understood from the results described in Table 2.

Also, non-patent document 2 describes an experiment conducted bymeasuring asialo AGP in sera of healthy subjects, acute hepatitispatients, chronic hepatitis patients, cirrhosis patients, and hepaticcell carcinoma patients by immunochromatography using RCA lectin, so asto reveal whether each liver disease could be determined to be positiveor not. However, the technique described in non-patent document 2 isunsatisfactory in view of liver disease detection performance, since thesensitivity of cirrhosis detection, for example, is about 88%, but thefalse positive rate for chronic hepatitis patients is 63%, as understoodfrom the results described in Table 2.

PRIOR ART DOCUMENTS Patent Documents

-   Patent document 1: JP Patent Publication (Kokai) No. 10-26622 A    (1998)-   Patent document 2: JP Patent Publication (Kokai) No. 8-184594 A    (1996)

Non-Patent Documents

-   Non-patent document 1: Ingvar Ryden et al., Diagnostic Accuracy of    α1-Acid Glycoprotein Fucosylation for Liver Cirrhosis in Patients    Undergoing Hepatic Biopsy, Clinical Chemistry 48: 12, 2195-2201    (2002)-   Non-patent document 2: Eun Young Lee et al., Development of a rapid,    immunochromatographic strip test for serum asialo glycoprotein in    patients with hepatic disease, Journal of Immunological Methods    308 (2006) 116-123

SUMMARY OF THE INVENTION

An object of the present invention is to provide a method for measuringa glycan-marker glycoprotein, by which liver disease can be detectedwith higher accuracy than conventional methods. Also, an object of thepresent invention is to provide a method for examining liver disease, bywhich liver disease can be detected with higher accuracy thanconventional methods. Also, an object of the present invention is toprovide a reagent(s) for quantitative determination of a glycoprotein(s)to be used for the above measurement methods. Moreover, an object of thepresent invention is to provide a glycan-marker glycoprotein as an indexfor clinical conditions of liver disease, with which clinical conditionsof liver disease can be distinguished from one another according to theprogress of liver disease.

The method for measuring at least one glycoprotein selected fromalpha-1-acid glycoprotein (AGP) and Mac-2-binding protein (M2BP)contained in a sample collected from a subject is characterized in that:when the glycoprotein is AGP, AGP that binds to first lectin selectedfrom AOL and MAL is measured; and when the glycoprotein is M2BP, M2BPthat binds to second lectin selected from WFA, BPL, AAL, RCA120, andTJAII is measured.

According to the measurement method of the present invention, aglycan-marker glycoprotein, with which liver disease such as cirrhosiscan be examined with high reliability, can be conveniently measured.

Furthermore, according to the glycan-marker glycoprotein as an index forclinical conditions of liver disease of the present invention,examination becomes possible with higher accuracy than is possible withexisting markers and with minute amounts of serum. Thus, monitoring ofthe progress of liver fiber formation becomes possible, so that not onlythe understanding of the development of clinical conditions but also theevaluation of improvement in fiber formation in the liver or liverinflammation after antiviral therapy using interferon, for example,become possible.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the time course from infection with type C hepatitis tohepatic cell carcinoma and changes in liver status.

FIG. 2 shows carcinogenic rates (from chronic hepatitis to hepatic cellcarcinoma).

FIG. 3 shows carcinogenic rates (from cirrhosis to hepatic cellcarcinoma).

FIG. 4 shows the relationship between changes in background liver tissueand carcinogenesis.

FIG. 5 shows structural changes in the liver from infection tocarcinogenesis.

FIG. 6 shows the relationship between changes in liver status anddiagnostic treatment schemes.

FIG. 7 shows experimental procedures for verification of candidatebiomarker molecules based on a lectin microarray.

FIG. 8 shows the results of conducting differential glycan analysis foral acid glycoprotein (AGP) that is one of marker glycoproteins asindices for clinical conditions of liver disease using antibody-overlaylectin microarray.

FIG. 9 shows the results of conducting differential glycan analysis for90K/Mac-2 Binding Protein (M2BP) that is one of marker glycoproteins asindices for clinical conditions of liver disease using anantibody-overlay lectin microarray.

FIGS. 10A and 10B show the correlations between the progress of fiberformation in the liver and changes in lectin signal intensity obtainedby antibody-overlay lectin array analysis of AGP.

FIG. 11 shows detection schemes for cirrhosis by antibody-overlay lectinmicroarray analysis of AGP that is a glycan marker as an index forclinical conditions of liver disease.

FIG. 12 shows the determination of therapeutic effects of interferonusing AGP as a marker capable of monitoring the progress of fiberformation in the liver.

FIG. 13 shows dilution linearity when DSA was used in a 1^(st) rapidmeasurement method.

FIG. 14 shows dilution linearity when MAL was used in the 1^(st) rapidmeasurement method.

FIG. 15 shows dilution linearity when AOL was used in the 1^(st) rapidmeasurement method.

FIG. 16 shows measurement results when MAL was used for commercialspecimens in the 1st rapid measurement method.

FIG. 17 shows measurement results when AOL was used for commercialspecimens in the 1st rapid measurement method.

FIG. 18 shows measurement results when MAL was used for commercialspecimens in a lectin array method.

FIG. 19 shows measurement results when AOL was used for commercialspecimens in a lectin array method.

FIG. 20 shows dilution linearity when DSA was used in a 2^(nd) rapidmeasurement method.

FIG. 21 shows dilution linearity when MAL was used in the 2^(nd) rapidmeasurement method.

FIG. 22 shows dilution linearity when AOL was used in the 2^(nd) rapidmeasurement method.

FIG. 23 shows measurement results when MAL was used for commercialspecimens in the 2^(nd) rapid measurement method.

FIG. 24 shows measurement results when AOL was used for commercialspecimens in the 2^(nd) rapid measurement method.

FIG. 25 shows measurement results when MAL was used for clinicalspecimens in the 2^(nd) rapid measurement method.

FIG. 26 shows measurement results when AOL was used for clinicalspecimens in the 2^(nd) rapid measurement method.

FIG. 27 shows measurement results when MAL was used for clinicalspecimens in a lectin array method.

FIG. 28 shows measurement results when AOL was used for clinicalspecimens in the lectin array method.

FIG. 29 shows the results of measuring sera of 125 cases of HCV-infectedpatients by the 2^(nd) rapid measurement method.

FIG. 30 shows comparison of the results obtained from 100 specimens ofhealthy subjects by a lectin array method with the results obtained fromthe same by the 2^(nd) rapid measurement method.

FIG. 31 shows correlation between the progress of fiber formation in theliver and changes in lectin signal intensity obtained byantibody-overlay lectin array analysis of M2BP.

FIG. 32 shows preoperative and postoperative glyco-alterations in M2BPin sera from hepatic cell carcinoma patients.

FIG. 33 shows a lectin-antibody sandwich ELISA model that is the bestmode for detection of WFA-binding M2BP.

FIG. 34 shows the postoperative time course changes in the amounts ofWFA-binding M2BP in sera from liver cancer patients.

FIG. 35 shows the effects of the presence or the absence of heattreatment for specimens on detection sensitivity during detection ofWFA-binding M2BP by ELISA.

FIG. 36 shows the dilution linearity of a measurement system using WFAwhen the supernatants of cultured HepG2 cells were used as samples inmeasurement of M2BP by the 2^(nd) rapid measurement method.

FIG. 37 shows the dilution linearity of a measurement system using WFAwhen recombinant human galectin-3BP/MAC-2BP was used as a sample inmeasurement of M2BP by the 2^(nd) rapid measurement method.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 1. Current Status ofChronic Liver Disease 1-1. Pathological Conditions of Liver Disease

After infection with hepatitis type B virus or hepatitis type C virus,transition from acute-stage inflammation to chronic-stage inflammationtakes place for 5-15 years. In particular, type C hepatitis that hastransitioned to the chronic stage is merely cured naturally, and theliver functions decrease to result in cirrhosis. The time course frominfection with type C hepatitis to hepatic cell carcinoma and changes inliver status are shown in FIG. 1. For definition of clinical conditionsfrom chronic hepatitis to cirrhosis, fibrous alterations that appear inthe hepatic region of Glisson's capsule and hepatic lobules are capturedpathomorphologically so that the disease is classified into the mild(F1), moderate (F2), severe (F3), or cirrhosis stage (F4). The progressof fiber formation correlates with increased risk of hepatic cellcarcinoma. As shown in FIG. 2, whereas the annual rate is 1% or less inthe case of F1 or F2, the annual rate increases to 3-4% in the case ofF3. In the case of cirrhosis (F4) diagnosed through confirmation of atissue image showing the further progress of fiber formation, hepaticcell carcinoma appears at an annual rate of about 7% as shown in FIG. 3.Therefore, it is particularly important for efficient detection andtreatment of hepatic cell carcinoma to conveniently select patients atthe F3 stage and the F4 stage and conduct follow-ups for such subjectsto be subjected to detailed examination.

The medical benefits project in Japan for type B and type C hepatitispatients are exclusively intended for fiber formation at the stagesranging from F1 to F3 as determined by histopathological diagnosis onliver biopsy specimens. On the other hand, when diseases are diagnosedat the F4 stage, they are classified as cirrhosis. Hence, only some typeB and type C hepatitis patients are aided by interferon treatment in themedical benefits project. In such cases, satisfactory treatment resultscannot be obtained.

1-2. Evaluation of Suppression of Fiber Formation by Antiviral Therapy

In the case of type C chronic hepatitis, PEG-IFN+RBV therapy is applied.In the case of a type C cirrhosis compensatory period, soleadministration of interferon is applied. Meanwhile, in the case of typeB hepatitis (chronic hepatitis or cirrhosis), treatment is mainlyperformed with a nucleic acid analog and inflammation and fiberformation evaluation markers are thought to be essential. In particular,clinical application of serum biomarkers is broadly expected fordiagnostic and evaluation purposes.

1-3. Hepatic Cell Carcinoma

It is considered that microbiological factors (through infection withhepatitis type B virus or hepatitis type C virus) and environmentalfactors alternately have significant impact on hepatic cell carcinoma.In Japan, it is known that about 90% of hepatic cell carcinoma patientshave a history of infection with hepatitis type B or hepatitis type Cvirus and that chronic hepatitis patients and cirrhosis patients develophepatic cell carcinoma. In addition to the presence of viruses, beingmale, being elderly, alcoholism, tobacco, the presence of aflatoxin (afungal toxin), and the like are suggested as hepatic cell carcinoma riskfactors (Clinical guidelines for liver cancer, International MedicalInformation Center (Foundation)).

1-4. Early Diagnosis of Hepatic Cell Carcinoma

Hepatic cell carcinoma is currently detected by mainly the measurementof liver cancer markers such as AFP and PIVKA-II in a serum sample froma subject and diagnostic imaging performed mainly by ultrasonography(echo test). Diagnostic imaging is generally performed by a first testusing ultrasonography or CT and then by MRI or angiography when someabnormalities are found.

1-5. Discrimination of Groups at High Risk of Cancer in View ofPrevention of Hepatic Cell Carcinoma

About 90% of hepatic cell carcinoma patients have experienced transitionfrom hepatitis due to infection with hepatitis type B virus or hepatitistype C virus in Japan. Hence, patients to be subjected to detailedexamination can be discriminated using viral infection and decreasedliver functions as indices.

However, even in the case of cirrhosis (F4) patients for which hepaticcell carcinoma appears at an annual rate of about 7%, it must be saidthat repetition of expensive detailed examinations that are highlyinvasive every 3 months for early detection and treatment of cancer is asignificant economic and physical burden for the patients. This canespecially be said for the case of F3, which has an annual carcinogenicrate of 3-4%. Furthermore, in view of the about 50% success rate of thetreatment of hepatitis type C virus with interferon, it is necessary toclarify the stage (between cirrhosis and the onset of hepatic cellcarcinoma) of each chronic hepatitis patient and thus to performclinical follow-up for a considerable number of the patients for whichinterferon treatment has failed. Specifically, current treatment for thedisease that proceeds from hepatitis to hepatic cell carcinoma is in astatus in which the risk of cancer should be ranked for hepatitis tocirrhosis patients by a convenient test such as a blood test and thendiagnostic treatment of hepatic cell carcinoma appropriate for eachresult should be performed.

From the perspective of clinical pathology, the degree of fiberformation correlates with the risk of cirrhosis or the risk of hepaticcell carcinoma. Therefore, we have found that the development of aninspection technique by which the progress of fiber formation can bedetermined serologically and quantitatively can solve the problem. Asshown in FIG. 4, infection with hepatitis type C virus that occurs inthe liver induces fiber formation in order to regenerate damagedhepatocytes and repair cicatrices. The risk of cancer increases as fiberformation in the liver proceeds. Hence, “the degree of fiber formation”can be used as an index for the onset of cancer. In background livertissue in which cancer develops, component cells are changed.Accordingly, it is inferred that glyco-alterations take place as fiberformation proceeds. As shown in FIG. 5, infection leads to the onset ofcancer with time. At this time, the loss of constant activity such asthe loss of normal structures and the loss of normal functions isobserved in the liver simultaneously with the advent of pathologicalstructures characterized by fiber formation. Hepatic cell carcinoma isknown to undergo transition from early hepatic cell carcinoma toclassical hepatic cell carcinoma, such that the properties of the cellsare altered. In cases of early hepatic cell carcinoma of a size oflarger than 2 cm, classical hepatic cell carcinoma appears. FIG. 6 showsthe relationship between changes in liver status and diagnostictreatment schemes. Whereas peginterferon/ribavirin combination therapy(PEG-IFN+RBV therapy) is performed for chronic hepatitis, radiowavecauterization therapy (RFA) is performed for early hepatic cellcarcinoma. There are neither diagnostic testing methods nor effectivetherapeutic methods for cirrhosis. The glycan-marker glycoproteins ofthe present invention can distinguish among chronic hepatitis,cirrhosis, and hepatic cell carcinoma, and thus can be used as an indexin the development of new treatment for cirrhosis. Also, theglycan-marker glycoproteins are expected to enable quantitativeevaluation of fiber formation through use in combination with theFibroscan. The glycan-marker glycoproteins also make it possible todiscriminate cases with fiber formation (F3-4). The glycan-markerglycoproteins are expected to be used as serum evaluation markers forevaluation of therapeutic effects, in addition to the quantitativediagnosis of fiber formation, upon clinical introduction of thetreatment of the progress of fiber formation in the liver or suppressionof the onset of cancer.

2. Liver-Disease-Specific Glyco-Alterations in New Glycan-MarkerGlycoproteins as Indices for Clinical Conditions of Liver Disease

AGP and M2BP are the new glycan-marker glycoproteins used as indices forclinical conditions of liver disease of the present invention. They arefound to experience glyco-alterations that are characteristic of chronichepatitis, cirrhosis, and hepatic cell carcinoma caused by thedevelopment and progress of diseases due to viral infection. Such markerglycoproteins capable of specifying clinical conditions of liver diseaseusing glyco-alterations accompanying the progress of clinical conditionsof viral liver disease as indices are referred to as glycan markers asindices for clinical conditions of liver disease.

Detection of glyco-alterations in AGP and M2BP that are new glycanmarkers as indices for clinical conditions of liver disease of thepresent invention is effective for distinguishing among clinicalconditions of viral liver diseases such as hepatic cell carcinoma,cirrhosis, fiber formation in the liver (F3 and F4 markers), and chronichepatitis so as to distinguish among the diseases. However, suchglyco-alterations in these markers differ in type and rate depending ondisease type and the degree of progress. Therefore, lectin probesreflecting disease-specific glyco-alterations should be statisticallyselected in order to construct detection kits specialized in eachsubject using the markers.

3. Verification of Liver Disease-Specific Glyco-Alterations inGlycan-Marker Glycoproteins

Disease-specific glyco-alterations existing on AGP and M2BP are verifiedusing lectin arrays described later. More specifically, anantibody-overlay lectin array method is employed. Based on the resultsobtained by measurement of the above marker glycoproteins using lectinarrays, 1) the degree of changes in measured values and lectin signalsat which the changes are observed depending on the progress of thedisease, 2) the stage (initial or late stage) at which changes inmeasured value are most significant, and 3) whether or not informationconcerning changes in measured value contributes to disease control areexamined and the usefulness thereof is evaluated, so that theappropriateness of each marker for clinical conditions of liver diseaseis verified.

More specific methods for selecting lectin probes appropriate for usingglycan-marker glycoproteins for monitoring of the progress of fiberformation in the liver or detection of cirrhosis are as described below.

Differential glycan profiling is performed using antibody-overlay lectinmicroarrays or the like for glycoproteins (AGP and M2BP) collected fromthe sera of (viral) hepatitis patients, cirrhosis patients, and hepaticcell carcinoma patients. First, blood samples are collected from (viral)hepatitis patients, chronic hepatitis patients, cirrhosis patients, andhepatic cell carcinoma patients. Each blood sample collected issubjected to immunoprecipitation using antibodies against AGP and M2BP,thereby performing concentration and purification. Then, it is confirmedwhether the use as glycan-marker glycoproteins for clinical conditionsof liver disease is possible using antibody-overlay lectin arrays. Morespecifically, as shown in FIG. 7, sera from patients with hepatitisviruses are subjected to differential glycan analysis for AGP and M2BP.AGP and M2BP are each simply enriched by an immunoprecipitation methodusing an anti-AGP antibody and an anti-M2BP antibody. A lectinmicroarray is a highly sensitive apparatus for differential glycananalysis. With such a lectin microarray, analysis can be sufficientlyconducted with about 100 nanograms of a protein (prepared). Hence, theabove pre-treatment can be performed on a small scale. Enriched AGP andM2BP are immediately added to a lectin microarray. After a givenreaction time, the sugar chain profiles of AGP and M2BP are obtained byan antibody-overlay lectin microarray method. At this time, the amountof a protein to be added to a lectin array differs depending on proteinand ranges from about several nanogram to several tens of nanograms.Array analysis is conducted for a sufficient number of specimens toallow statistical analysis, and then 2-group comparison analysis (e.g.,Student-T test) is conducted using the resulting data set. Therefore,objective selection of lectins (which can cause significant differencesin signals resulting from changes in clinical conditions) becomespossible. As a lectin microarray, for example, a lectin microarray uponwhich a plurality of lectins including some or all of the lectins listedin Table 2 shown below have been immobilized can be used. Morespecifically, lectin microarrays described in the document of Kuno A.,et al., Nat. Methods 2, 851-856 (2005) or LecChip (GP Bioscience) can beused. As antibodies, antibodies listed in Table 1 can be used.

TABLE 1 Glycoprotein name Antibody (Bender, catalog No.) alpha-1-acidglycoprotein (AGP) mouse monoclonal, clone AGP-47 (Sigma, A5566) rabbitpolyclonal (Rockland, 200-406-046) Galectin 3 binding protein goatpolyclonal (R&D Systems, (Mac-2-binding protein, Mac2BP) AF2226

3-1. Lectin Microarray (Also Simply Referred to as Lectin Array)

A lectin array is prepared by immobilizing (array formation) a pluralityof types of discriminant (probe) lectin, which differ in specificity, onone substrate in parallel. With a lectin array, the types of lectin thatinteract with complex carbohydrates to be analyzed and the degrees ofthe interactions can be simultaneously analyzed. Moreover, with a lectinarray, information required for glycan structure estimation can beobtained by a single analysis, and, steps from sample preparation toscanning can be rapidly and conveniently performed. Glycoproteins cannotbe directly analyzed by a glycan profiling system such as massspectroscopy. When such a system is employed, glycoproteins should beconverted in advance to glycopeptides or free sugar chains. Meanwhile, alectin microarray is advantageous in that direct analysis is possiblewith only the direct introduction of a fluorescence substance into acore protein portion, for example. Lectin microarray technology has beendeveloped by the present inventors and the principle and the basicconcept thereof are described in Kuno A., et al. Nat. Methods 2, 851-856(2005), for example.

Examples of lectins to be used for lectin arrays are as listed in Table2 below.

TABLE 2 Binding specificity Lectins Origin (Sugar binding specificity) 1LTL Lotus tetragonolobus Fucα1-3GlcNAc, Sia-Le^(x) and Le^(x) 2 PSAPisum sativum Fucα1-6GlcNAc and α-Man 3 LCA Lens culinaris Fucα1-6GlcNAcand α-Man, α-Glc 4 UEA-I Ulex europaeus Fucα1-2LacNAc 5 AOL Aspergillusoryzae Terminal αFuc and ±Sia-Le^(x) 6 AAL Aleuria aurantia TerminalαFuc and ±Sia-Le^(x) 7 MAL Maackia amurensis Siaα 2-3Gal 8 SNA Sambucusnigra Siaα 2-6Gal/GalNAc 9 SSA Sambucus sieboldiana Siaα 2-6Gal/GalNAc10 TJA-I Trichosanthes japonica Siaα 2-6Galβ1-4GlcNAcβ-R 11 PHA(L)Phaseolus vulgaris Tri- and tetra-antennary complex oligosaccharides 12ECA Erythrina cristagalli Lac/LacNAc 13 RCA120 Ricinus communisLac/LacNAc 14 PHA(E) Phaseolus vulgaris NA2 and bisecting GlcNAc 15 DSADatura stramonium (GlcNAc)_(n), polyLacNAc and LacNAc (NA3, NA4) 16GSL-II Griffonia simplicifolia Agalactosylated N-glycan 17 NPA Narcissuspseudonarcissus non-substituted α1-6Man 18 ConA Canavalia ensiformisα-Man (inhibited by presence of bisecting GlcNAc) 19 GNA Galanthusnivalis non-substituted α1-6Man 20 HHL Hippeastrum hybridnon-substituted α1-6Man 21 BPL Bauhinia purpurea alba Galβ1-3GalNAc andNA3, NA4 22 TJA-II Trichosanthes japonica Fucα1-2Gal, β-GalNAc > NA3,NA4 23 EEL Euonymus europaeus Galα1-3[Fucα1-2Gal] > Galα1-3Gal 24 ABAAgaricus bisporus Galβ1-3GalNAcα-Thr/Ser (T) and sialyl-T 25 LELLycopersicon esculentum (GlcNAc)_(n) and polyLacNAc 26 STL Solanumtuberosum (GlcNAc)_(n) and polyLacNAc 27 UDA Urtica dioica (GlcNAc)_(n)and polyLacNAc 28 PWM Phytolacca americana (GlcNAc)_(n) and polyLacNAc29 Jacalin Artocarpus integrifolia Galβ1-3GalNAcα-Thr/Ser (T) andGalNAcα-Thr/Ser (Tn) 30 PNA Arachis hypogaea Galβ1-3GalNAcα-Thr/Ser (T)31 WFA Wisteria floribunda Terminal GalNAc (e.g., GalNAcβ1-4GlcNAc) 32ACA Amaranthus caudatus Galβ1-3GalNAcα-Thr/Ser (T) 33 MPA Maclurapomifera Galβ1-3GalNAcα-Thr/Ser (T) and GalNAcα-Thr/Ser (Tn) 34 HPAHelix pomatia Terminal GalNAc 35 VVA Vicia villosa α-,β-linked terminalGalNAc and GalNAcα-Thr/Ser (Tn) 36 DBA Dolichos biflorus GalNAcα-Thr/Ser(Tn) and GalNAcα1-3GalNAc 37 SBA Glycine max Terminal GalNAc (especiallyGalNAcα1-3Gal) 38 GSL-I Griffonia simplicifolia α-GalNAc,GalNAcα-Thr/Ser (Tn), α-Gal mixture 39 PTL-I Psophocarpus α-GalNAc andGal tetragonolobus 40 MAH Maackia amurensis Siaα2-3Galβ1-3[Siaα2-6GalNAc]α-R 41 WGA Triticum unigaris (GlcNAc)n andmultivalent Sia 42 GSL-IA₄ Griffonia simplicifolia α-GalNAc,GalNAcα-Thr/Ser (Tn) 43 GSL-IB₄ Griffonia simplicifolia α-Gal

For example, a lectin array (LecChip (GP Bioscience)) on which 45 typesof lectin have been immobilized on the base is already currentlycommercially available.

3-2. Statistical Analysis of Glycan Profiles Using Lectin Arrays

Lectin array technology has been currently developed to practicaltechnology by which quantitative differential glycan profiling can beperformed not only for purified samples, but also for mixed samples suchas sera and cell lysates. In particular, differential glycan profilingof sugar chains on cell surface layers has been significantly developed(Ebe, Y. et al., J. Biochem. 139, 323-327 (2006), Pilobello, K. T. etal., Proc Natl Acad Sci U.S.A. 104, 11534-11539 (2007), Tateno, H. etal., Glycobiology 17, 1138-1146 (2007)).

Also, data mining of glycan profiles by statistical analysis can beperformed by methods described in “Kuno A, et al. J ProteomicsBioinform. 1, 68-72 (2008),” “The Japanese Society of CarbohydrateResearch, 2008/8/18 Development of Applied Technology of LectinMicroarray—Differential Glycan Profiling and Statistical Analysis ofBiological Samples—Atsushi Kuno, Atsushi Matsuda, Yoko Itakura, HidekiMatsuzaki, Hisashi Narumatsu, and Jun Hirabayashi”, and “Matsuda A, etal. Biochem Biophys Res Commun. 370, 259-263 (2008),” for example.

3-3. Antibody-Overlay Lectin Microarray Method

A lectin microarray platform is basically as described above. Theantibody-overlay lectin microarray method is an applied method thatenables convenient, simultaneous, and high-speed analysis of multipleanalytes upon detection not by directly labeling analytes withfluorescence or the like, but by indirectly introducing a fluorescentgroup or the like via an antibody into analytes (“Kuno A, Kato Y,Matsuda A, Kaneko M K, Ito H, Amano K, Chiba Y, Narimatsu H, HirabayashiJ. Mol Cell Proteomics. 8, 99-108 (2009),” “Jun Hirabayashi, AtsushiKuno, and Noboru Uchiyama, “Development of Applied Technology of GlycanProfiling using Lectin Microarray,” “Approach from Molecular Level,Cancer Diagnosis and Research—Challenge to Clinical Application—(ExtraNumber, Experimental Medicine), YODOSHA, Vol. 25 (17) 164-171 (2007),”“Application of Glycan Profiling System using Lectin Microarray toSearch for Glycan marker (Atsushi Kuno and Jun Hirabayashi),” and“Development of Clinical Glycan marker and Elucidation of Sugar ChainFunctions (see Gene and Medicien, Mook No. 11, pp. 34-39, Medical Do(2008)).

When glycoproteins (AGP and M2BP) are analytes, sugar chain (glycan)portions are recognized by lectins on a lectin microarray. Antibodies(anti-AGP antibody and anti-M2BP antibody) against core protein portionsare overlaid thereon, so that the glycoproteins to be tested can bespecifically detected with high sensitivity without labeling orhigh-level precise purification thereof.

3-4. Lectin-Overlay Antibody Microarray Method

The lectin-overlay antibody microarray method involves the use of anantibody microarray instead of a lectin microarray, which is prepared byimmobilizing in parallel (array formation) an antibody against a coreprotein on a substrate such as a glass substrate. The number ofantibodies corresponding to the number of markers to be examined isrequired for the method. It is also required to confirm in advancelectins for detection of glyco-alterations.

4. Method for Detecting Liver Disease Using Disease-SpecificGlyco-Alterations in Glycan Marker as an Index for Clinical Conditionsof Liver Disease

AGP and M2BP are novel glycan markers as indices for clinical conditionsof liver disease, which is characterized in that the glycan structuresare altered in association with clinical changes in liver disease suchas the progress of fiber formation. Accordingly, a lectin (hereinafter,abbreviated as lectin “A”), the reactivity of which is altered inresponse to changes in the glycan structures of AGP and M2BP is reactedwith markers contained in a sample collected from a subject, and thenmarkers that have reacted with the lectin are measured. Thus, theclinical conditions of liver disease can be identified and the degree offiber formation in the liver can be determined, for example.

For example, novel glycan markers as indices for clinical conditions ofliver disease can be detected using:

(1) (i) the above lectin “A”; and (ii) an antibody for detection of acore protein portion other than the sugar chain of the above marker.Novel glycan markers as indices for clinical conditions of liver diseasecan also be detected using: (2) antibodies that are specific to glycanmarkers as indices for clinical conditions of liver disease, the epitopeof which is a part containing a sugar-chain-binding portion.

For example, markers are detected using antibodies against the coreproteins of markers and lectin “A”, so that liver disease patients canbe distinguished from healthy subjects and detected. Preferably, anantibody overlay method using lectin array (“Kuno A, Kato Y, Matsuda A,Kaneko M K, Ito H, Amano K, Chiba Y, Narimatsu H, Hirabayashi J. MolCell Proteomics. 8, 99-108 (2009)) can be used. When one, two, or moreoptimum lectins “A” are selected for the test for verification ofdisease-specific glyco-alterations in 3, it is more preferable to use a1^(st) or 2^(nd) rapid measurement method (described later).

A specific example of the method for examining liver disease using novelglycan markers as indices for clinical conditions of liver disease is amethod for detecting liver disease comprising the steps of:

1) measuring glycan markers as indices for clinical conditions of liverdisease, which have sugar chains specifically reacting with lectin “A”in a sample collected from a subject;

2) measuring glycan markers as indices for clinical conditions of liverdisease, which have sugar chains specifically reacting with lectin “A”in a sample collected from a healthy subject;

3) measuring glycan markers as indices for clinical conditions of liverdisease, which have sugar chains specifically reacting with lectin “A”in a sample collected from a liver disease patient; and

4) comparing the measurement results for glycan markers as indices forclinical conditions of liver disease obtained from the subject with themeasurement results for glycan markers as indices for clinicalconditions of liver disease obtained from the healthy subject or theliver disease patient, and then determining that the subject has liverdisease when the measurement results for the subject are closer to themeasurement results for the liver disease patient.

A threshold is also determined in advance for distinguishing liverdisease from the other diseases based on the measurement results formany liver disease patients and healthy subjects, and then themeasurement results for the subject are compared with the threshold, soas to determine whether or not the subject is affected by liver disease.

4-1. Method for Measuring the Progress of Fiber Formation

It is known that regarding the progress of hepatitis due to hepatitisviral infection, the degree of fiber formation correlates with liverdysfunction and a risk of hepatic cell carcinoma. Therefore, themeasurement of fiber formation refers to evaluation of liver dysfunctionand a risk of cancer. Also, about 40% of all the hepatitis patients doesnot react to interferon treatment, so that viral infection persists. Itis considered that whether or not these clinical conditions progress toan active mode should be determined based on the progress of fiberformation. Based on these viewpoints, measurement of the progress offiber formation is significant for diagnostic treatment for hepatitis.

Fiber formation is currently evaluated based on pathological diagnosison biopsy samples. In recent years, the widespread use of the method isexpected as a result of introduction of Fibroscan. Also, as methods forserological evaluation of fiber formation, Fibro Test, Form's index,Hepatoscore, and the like are clinically used, but these methods areinferior to biopsy diagnosis in terms of both sensitivity andspecificity.

AGP and M2BP are subjected to the antibody-overlay lectin microarraymethod using a group of patients' sera differing in the degree of theprogress of fiber formation, so that lectins that exhibit increased ordecreased signal intensity in correlation with the degree of theprogress of fiber formation are selected. Based on the information, asandwich detection method using an antibody against a candidate markermolecule and lectin “A” exhibiting changes in signals due to theprogress of fiber formation can be established, such as lectin-antibodysandwich ELISA or an antibody-overlay lectin microarray method. About100 patients' sera (100 serum samples) subjected in advance to thestaging of fiber formation by pathological diagnosis are collected andthen subjected to analysis. A cut off value at each stage is thendetermined, so that the progress of fiber formation in the liver can bemonitored using the serum sample from each patient.

An example of lectin “A” is, in the case of AGP, a first lectin selectedfrom AOL and MAL and an example of lectin “A” in the case of M2BP is asecond lectin selected from WFA, BPL, AAL, RCA120, and TJAII.Hereinafter, such a lectin selected from AOL and MAL may be referred toas a “first lectin,” and such a lectin selected from WFA, BPL, AAL,RCA120, and TJAII may be referred to as a “second lectin.”

For example, AGP binding to a first lectin can be measured using a firstlectin array to which at least the first lectin has been immobilized andan anti-AGP antibody; and M2BP binding to a second lectin can bemeasured using a second lectin array to which at least the second lectinhas been immobilized and an anti-M2BP antibody.

4-2. Detection of Cirrhosis

Cirrhosis is defined as clinical conditions in which regeneratingnodules losing hepatic lobule structures and fine fibrous diffuseconnective tissues appear throughout the liver, surrounding suchregenerating nodules. This is also a terminal status of progressivechronic liver disease with persistent damage to hepatocytes and fiberformation. Liver biopsy to be performed in cirrhosis is intended toperform component diagnosis. In the cases of early cirrhosis orcirrhosis with a macronodular pattern, many cases are diagnosed withdifficulties (Surgical Pathology, Fourth ed., Bunkodo Co., Ltd.).Therefore, test techniques with which cirrhosis can be qualitatively andquantitatively diagnosed are required. For the purpose, candidateantibody molecules capable of monitoring the progress of fiber formationand a lectin set, found in the section, “1) Method for measuring theprogress of fiber formation” can be used for detection of cirrhosis ifthe fiber formation stages, F3 and F4, can be distinguished from eachother.

4-3. Detection of Disease-Specific Glyco-Alterations on Novel GlycanMarkers as Indices for Clinical Conditions of Liver Disease in a Sample

Examples of a sample include, biopsy tissue samples, body fluid samples,and a preferable example of the same is blood (e.g., serum and bloodplasma).

The term “measurement” refers to both qualitative measurement andquantitative measurement.

Measurement of glycan markers as indices for clinical conditions ofliver disease can be performed using (1) a column or an array to whichlectin “A” has been immobilized and (2) an antibody against AGP or M2BP,for example. Preferably, an antibody-overlay lectin array method, andmore preferably a 1^(st) or 2^(nd) rapid measurement method can be used.

The concentration of AGP or M2BP can also be measured. Examples of amethod to be used therefor include an antibody-overlay lectin arraymethod using a lectin array, immunoassay, an enzyme activitydetermination method, and a capillary electrophoresis method.Preferably, the following qualitative or quantitative techniques can beused, such as enzyme immunoassay, double-antibody sandwich ELISA, a goldcolloid method, radioimmunoassay, enzyme chemiluminescence immunoassay,electric chemiluminescence immunoassay, latex agglutination immunoassay,fluorescence immunoassay, a Western blotting method, animmunohistochemical method, and a surface plasmon resonance method(hereinafter, referred to as an SPR method) using lectin “A” mostreflecting disease-specific glyco-alterations, which is statisticallyselected with an antibody-overlay lectin array, and a monoclonalantibody or a polyclonal antibody specific to AGP or M2BP.

Further specifically, sub determination can also be performed by aWestern blotting method using lectin “A” and antibodies against glycanmarkers as indices for clinical conditions of a disease. The aboveexpression “when the measurement results for a subject are higher than .. . ” in qualitative determination refers to a case in which novelglycan markers as indices for clinical conditions of a disease arequalitatively demonstrated to be present in a sample from a subject withhigher concentrations than those in a sample from a healthy subject.Furthermore, a lectin method as a direct sugar chain measurement methodwithout mediation of an antibody is also included herein.

Examples of lectin “A” the reactivity of which is altered in response tochanges (accompanying the progress of fiber formation in the liver) inthe AGP glycan structure include AOL and MAL or a combined use of AOLand MAL, which are strictly selected by statistical analysis after anantibody-overlay lectin array method. AOL is a lectin that exhibits thehighest significant difference among a group of lectins, the reactivityof which to AGP sugar chain increases with the progress of fiberformation in the liver. MAL is a lectin exhibits the most significantdifferences among a group of lectins, the reactivity of which to AGPsugar chain decreases with the progress of fiber formation in the liver.Accordingly, the use of both measured value of AGP binding to AOL andmeasured value of AGP binding to MAL makes it possible to more preciselydistinguish the clinical conditions of the liver disease of a subject.As a method that involves the use of both the measured value of AGPbinding to AOL (measured value of AOL) and the measured value of AGP(measured value of MAL) binding to MAL, a known statistical techniquecan be employed. For convenience, a difference between and the ratio ofAOL measurement value and/to MAL measurement value can be used. Inaddition, when a difference between or the ratio of AOL measurementvalue and/to MAL measurement value is used, the same scale is preferablyemployed for both measured values for convenience, a difference betweenthe cut off line values of both measured values is found and then thescales can be corrected. For example, the ratio of MAL cut-off linevalue to AOL cut-off line value is used and then either AOL measurementvalue or MAL measurement value may be corrected. AGP contained in asample collected from a subject using AOL and/or MAL is measured, sothat AGP can be used as a marker for identifying fiber formation in theliver, a cirrhosis detection marker, or a hepatic cell carcinomadetection marker. Also, AGP in a sample collected from a patient undertreatment using interferon or the like is measured using the lectin “A,”so that the therapeutic effect can be monitored.

In addition, the measured values of AGP binding to AOL or MAL arepreferably normalized using the measured value of AGP binding to lectinsthe reactivity of which substantially remains unchanged regardless ofchanges in AGP glycan structure. As such a lectin, DSA is preferable.Also, measured values of AGP binding to AOL or MAL can be normalizedusing the measured value of AGP core protein contained in a samplecollected from a subject. Also, in a method that involves the use ofboth AOL measurement value and MAL measurement value, a normalized AOLmeasurement value and a normalized MAL measurement value are preferablyused. For example, with the use of the above first lectin array and theabove second lectin array, which are prepared by further immobilizingDSA, AGP binding to DSA is measured, measured values of AGP binding to afirst lectin are normalized using measured values of AGP binding to DSA,M2BP binding to DSA is measured, the measured values of M2BP binding toa second lectin are normalized using the measured value of M2BP bindingto DSA, and thus AGP and/or M2BP can be measured. Examples of lectin“A,” the reactivity of which is altered in response to changes in M2BPglycan structure accompanying changes in the clinical conditions of theliver, include WFA, BPL, AAL, RCA120, and/or TJAII strictly selected bystatistical analysis after the antibody-overlay lectin array method.WFA, BPL, AAL, RCA 120, and TJAII are all lectins, the reactivity ofwhich to M2BP sugar chain increases as the clinical conditions of liverdisease progress. WFA, BPL, and TJAII that are strongly reactive to M2BPof hepatic cell carcinoma patients, and particularly of cases ofpost-operative cancer recurrence. M2BP contained in a sample collectedfrom a subject is measured using WFA, BPL and/or TJAII, so that M2BP canbe used as a marker for discriminating a group of patients with a highrisk of hepatic cell carcinoma. Also, M2BP contained in a samplecollected from a subject is measured using AAL and/or RCA120, so thatM2BP can be used as a marker for identifying fiber formation in theliver, a cirrhosis detection marker, or a hepatic cell carcinomadetection marker. Also, WFA is periodically used for a patient afterextraction of hepatic cell carcinoma and then M2BP in a sample collectedfrom the patient is measured, so that a risk of recurrence can bepredicted. In addition, there are very few molecules capable of bindingto WFA in glycoproteins contained in serum, thereby enabling the directuse of serum as a measurement sample. WFA is preferable since the degreeof freedom resulting from the use of WFA is high upon construction of ameasurement system.

In addition, the measured values of M2BP binding to WFA, BPL, AAL, RCA120, or TAJII are preferably normalized using the measured value of M2BPbinding to a lectin the reactivity of which substantially remainsunchanged regardless of changes in M2BP glycan structure. As such alectin, DSA is preferable. Also, the measured values of M2BP binding toWFA, BPL, AAL, RCA120, or TAJII may be normalized using the measuredvalue of M2BP core protein contained in a sample collected from asubject.

Measurement of AGP binding to AOL, MAL, or DSA and measurement of M2BPbinding to WFA, BPL, AAL, RCA120, or TAJII are preferably performed bythe antibody-overlay lectin microarray method. In particular, theantibody-overlay lectin microarray method is capable of simultaneouslymeasuring AGP or M2BP binding to a plurality of types of lectin. Also,when markers such as AGP and M2BP are rapidly measured using lectin “A,”measurement is preferably performed by the following 1^(st) or 2^(nd)rapid measurement method.

An example of the 1st rapid measurement method is a method forquantitatively determining AGP reacting with the above lectin “A” bymixing biotinylated lectin “A” prepared by binding biotin to lectin “A”with a sample, adding magnetic particles to which streptavidin has beenimmobilized to the mixture, so as to form a magnetic particle-lectin“A”-AGP complex, reacting the complex with a labeled anti-AGP antibody,so as to form a 2^(nd) complex of magnetic particle-lectin“A”-AGP-labeled anti-AGP antibody, and then measuring the amount of thelabel of the 2nd complex. In addition, M2BP can be measured in a mannersimilar to that in the above method for measuring AGP except for the useof a labeled anti-M2BP antibody. With the 1st rapid measurement method,AGP or M2BP in a sample can be quantitatively determined within about 60minutes. With the use of biotinylated lectin “A” andstreptavidin-immobilized magnetic particles, the reactivity of lectin“A” to AGP or M2BP in a sample can be improved and the reaction time oflectin “A” with AGP or M2BP can be shortened to about 30 minutes.

Furthermore, an example of the 2^(nd) rapid measurement method is amethod for quantitatively determining AGP reacting with lectin “A” bymixing magnetic particles to which lectin “A” has been immobilized witha sample, capturing AGP sugar chains in the sample using lectin “A,”reacting AGP captured by the magnetic particles with a labeled anti-AGPantibody, so as to form a magnetic particle-lectin “A”-AGP-labeledanti-AGP antibody complex, and then measuring the amount of the label ofthe complex. Also with the 2^(nd) rapid measurement method, M2BP can bemeasured in a manner similar to the above method for measuring AGPexcept for the use of a labeled anti-M2BP antibody. With the 2^(nd)rapid measurement method, AGP or M2BP in a sample can be quantitativelydetermined within about 20 minutes. Specifically with the use of lectin“A”-immobilized magnetic particles, the reactivity of lectin “A” to AGPor M2BP in a sample is significantly improved and thus the reaction timeof lectin “A” with AGP or M2BP can be shortened to about 5 minutes.

The 1^(st) and the 2^(nd) rapid measurement methods are suitable forautomation. In particular, the 2^(nd) rapid measurement method can beappropriately performed with full automatic measuring equipment.Automation of the 2nd rapid measurement method makes it possible toeasily perform consecutive measurement of multiple specimens. Also,automation makes it possible to perform measurement of a single specimenfor multi test items (measurements using different lectins) within ashort period of time.

As labels for labeled anti-AGP antibodies or labeled anti-M2BPantibodies, fluorescent substances or enzymes can be used. Examples of afluorescent substance include fluorescein isothiocyanate (FITC), a greenfluorescent protein (GFP), and luciferin. Examples of an enzyme includealkaline phosphatase (ALP), peroxidase, glucose oxidase, tyrosinase, andacid phosphatase. When alkaline phosphatase is used as an enzyme, aknown luminescent substrate, a known chromogenic substrate, and the likecan be used. Examples thereof include chemiluminescent substrates suchas CDP-star (registered trademark)(4-chloro-3-(methoxyspiro{1,2-dioxetane-3,2′-(5′-chloro)tricyclo[3.3.1.13,7]decane}-4-yl)phenylphosphatedisodium), and CSPD (registered trademark)(3-(4-methoxyspiro{1,2-dioxetane-3,2-(5′-chloro)tricyclo[3.3.1.13,7]decane}-4-yl)phenylphosphate disodium), and chromogenic substrates such asp-nitrophenyl phosphate, 5-bromo-4-chloro-3-indolyl-phosphoric acid(BCIP), 4-nitroblue tetrazolium chloride (NBT), and iodonitrotetrazolium(INT). Furthermore, an antibody is labeled with biotin, and then theabove fluorescent substance to which streptavidin has been bound or theabove enzyme may be bound to the antibody via biotin-avidin binding.

A blood sample contains a trace amount of AGP or M2BP having glycanstructural changes due to fiber formation in the liver. Hence, it ispreferable to use an enzyme as a label and a luminescent substrate inview of high sensitivity and more rapid detection of the label.

In addition, an enzyme such as ALP has sugar chains. Hence, adeglycosylated enzyme is preferably used to prevent a nonspecificreaction between the sugar chains and a lectin. As such a deglycosylatedenzyme, deglycosylated ALP can be used, such as Recombinant AP, EIADrade, and CR 03535452 (Roche Diagnostics).

Also, an anti-AGP antibody or an anti-M2BP antibody also has sugarchains. Hence, a deglycosylated antibody is preferably used to prevent anonspecific reaction between sugar chains and lectins. For example, ananti-AGP antibody or an anti-M2BP antibody converted to Fab′ as a resultof pepsin digestion and reduction is preferably used.

A labeled antibody reagent is prepared as follows. According to a knownmethod, an anti-AGP antibody or an anti-M2BP antibody is mixed with amaleimidized label using a cross-linking agent such as EMCS[N-(6-Maleimidocaproyloxy)succinimido] (DOJINDO LABORATORIES) forreaction, so that a labeled antibody can be prepared. For example,deglycosylated ALP is maleimidized using a cross-linking agent, followedby reaction with an anti-AGP antibody or an anti-M2BP antibody convertedto Fab′. The use of the thus prepared antibody is preferable in view ofprevention of nonspecific reaction.

5. Novel Specific Polyclonal Antibodies and/or Monoclonal AntibodiesUsing Novel Glycan Markers as Indices for Clinical Conditions of LiverDisease

In the method for detecting hepatic cell carcinoma using novel glycanmarkers as indices for clinical conditions of liver disease, when apolyclonal antibody and/or a monoclonal antibody specific to glycanmarkers as indices for clinical conditions of liver disease can beeasily obtained, these antibodies can be used. However, when theantibodies cannot be easily obtained, they can be prepared as follows,for example.

5-1. Preparation of Antibodies

The novel glycan markers as indices for clinical conditions of liverdisease of the present invention can be used for preparing a polyclonalantibody or a monoclonal antibody for detection of liver disease.

For example, antibodies against novel glycan markers as indices forclinical conditions of liver disease can be prepared by a known method.Freund's complete adjuvant is administered at the same time, so that thegeneration of an antibody can be boosted. Also, a peptide including abinding position to which sugar chain X has bound is synthesized, thepeptide is covalently bound to commercially available keyhole limpethemocyanin (KLH), and then the conjugate is administered to an animal.In addition, a granulocyte-macrophage colony stimulating factor (GM-CSF)is administered at the same time, so that antibody production can beboosted.

Also, for example, monoclonal antibodies against novel glycan markers asindices for clinical conditions of liver disease can be prepared by themethods of Keller and Milstein (Nature Vol. 256, pp. 495-497 (1975)).For example, a hybridoma is prepared by cell fusion ofantibody-producing cells obtained from an animal immunized with anantigen with myeloma cells, and then clones producing an anti-X antibodyare selected from the thus obtained hybridoma.

Specifically, an adjuvant is added to the thus obtained glycan markersas indices for clinical conditions of liver disease for antigens.Examples of an adjuvant include Freund's complete adjuvant and Freund'sincomplete adjuvant. Any of these adjuvants may be mixed.

An antigen obtained as described above is administered to a mammal suchas a mouse, a rat, a horse, a monkey, a rabbit, a goat, or sheep. Anyimmunization method can be employed herein, as long as it is an existingmethod. Immunization is mainly performed by intravenous injection,subcutaneous injection, intraperitoneal injection, or the like. Also,the immunization interval is not particularly limited and immunizationis performed at intervals of several days to several weeks andpreferably at intervals of 4 to 21 days.

On days 2 to 3 after the final immunization date, antibody-producingcells are collected. Examples of antibody-producing cells include spleencells, lymph node cells, and peripheral blood cells.

As myeloma cells to be fused to antibody-producing cells, cells ofestablished cell lines from various animals (e.g., mice, rats, andhumans), which are generally available for persons skilled in the art,are used. Cell lines to be used herein have properties such that theyhave drug resistance and are unable to survive in an unfused state, butare able to survive only in a fused state in a selective medium (e.g.,HAT medium). In general, an 8-azaguanine-resistant strain is used. Thiscell line is deficient in hypoxanthine-guanine-phosphoribosyltransferaseand is unable to grow in a hypoxanthine.aminopterin.thymidine (HAT)medium.

Myeloma cells of various known cell lines are appropriately used, suchas P3 (P3x63Ag8.653) (J. Immunol. 123, 1548-1550 (1979)), P3x63Ag8U.1(Current Topics in Microbiology and Immunology 81, 1-7 (1978)), NS-1(Kohler, G. and Milstein, C., Eur. J. Immunol. 6, 511-519 (1976)),MPC-11 (Margulies, D. H. et al., Cell 8, 405-415 (1976)), SP2/0(Shulman, M. et al., Nature 276, 269-270 (1978)), FO (de St. Groth, S.F. et al., J. Immunol. Methods 35, 1-21 (1980)), 5194 (Trowbridge, I.S., J. Exp. Med. 148, 313-323 (1978)), and 8210 (Galfre, G. et al.,Nature 277, 131-133 (1979)).

Next, the above myeloma cells are fused to antibody-producing cells.Cell fusion is performed by bringing myeloma cells into contact withantibody-producing cells at a mixture ratio ranging from 1:1 to 1:10 inthe presence of a fusion accelerator at 30° C. to 37° C. for 1 to 15minutes in a medium for culturing animal cells, such as MEM, DMEM, orRPME-1640 medium. A fusion accelerator with an average molecular weightbetween 1,000 and 6,000 such as polyethylene glycol or polyvinylalcohol, or a fusion virus such as Sendai virus can be used foracceleration of cell fusion. Also, antibody-producing cells can also befused to myeloma cells using a commercially available cell fusionapparatus using electrical stimulation (e.g., electroporation).

A hybridoma of interest is selected from cells after cell fusion. Anexample of a method for selection is a method using selective growth ofcells in a selective medium. Specifically, cell suspension is dilutedwith an appropriate medium and then spread over a microtiter plate. Aselective medium (e.g., HAT medium) is added to each well, and thencells are cultured while appropriately exchanging selective media. As aresult, cells that have grown can be obtained as hybridoma cells.

Screening for a hybridoma is performed by limiting dilution, afluorescence excitation cell sorter method, or the like, and thenfinally a monoclonal-antibody-producing hybridoma is obtained. Examplesof a method for collecting a monoclonal antibody from the thus obtainedhybridoma include a general cell culture method and a method for formingascites.

Also, an antibody to be used in the present invention may be amonoclonal antibody or a polyclonal antibody. Examples of such anantibody include single-chain Fvs (scFv), a single-chain antibody, a Fabfragment, a F(ab′) fragment, and disulfide linkage Fvs (sdFv).Furthermore, as an antibody to be used in not only the 1^(st) or the2^(nd) rapid measurement method, but also the present invention, adeglycosylated antibody is preferably used to prevent a nonspecificreaction between a sugar chain and a lectin. For example, an anti-AGPantibody or an anti-M2BP antibody converted to Fab′ as a result ofpepsin digestion and reduction can be used.

EXAMPLES Example 1 Use of Glycan-Marker Glycoproteins as Indices forClinical Conditions of Liver Disease for Detection of Liver Disease

Liver disease was detected by antibody-overlay lectin array performedfor glycan-marker glycoproteins as indices for clinical conditions ofliver disease, AGP and Mac2BP (M2BP), as follows. In addition, FIG. 7shows the procedures of this technique for differential analysis ofsugar chains on the marker glycoproteins derived from the sera of(viral) hepatitis patients (CH), cirrhosis patients (LC), hepatic cellcarcinoma patients (HCC), and healthy subjects (HV).

1. Enrichment of Marker Proteins from Serum

Enrichment of the marker glycoproteins derived from the sera of (viral)hepatitis patients (CH), cirrhosis patients (LC), hepatic cell carcinomapatients (HCC), and healthy subjects (HV) was performed according to“Kuno A, Kato Y, Matsuda A, Kaneko M K, Ito H, Amano K, Chiba Y,Narimatsu H, Hirabayashi J. Mol Cell Proteomics. 8, 99-108 (2009).” Inaddition, five cases each were used for analysis of each clinicalcondition in order to clarify the dependence of the obtained results onclinical conditions. Each patient's serum was diluted 10-fold with a0.2% SDS-containing PBS buffer and then heated for 10 minutes at 95° C.Five (5) μL of the resultant was dispensed in the case of AGP and 20 μLof the same was dispensed in the case of Mac2BP to reaction tubes, andthen 500 ng of an antibody (biotinylated antibody) against each antigenwas added to the reaction tubes. Each reaction solution was adjustedwith a reaction buffer (1% Triton X-100-containing Tris-buffered saline(TBSTx)) to 45 μL, followed by 2 hours of shaking reaction at 4° C.Immediately after antigen-antibody reaction, 5 μL (corresponding to 10μL of the original beads solution) of a solution of magnetic beads withstreptavidin immobilized thereto (Dynabeads MyOne Streptavidin T1, DYNALBiotech ASA), which had been washed 3 times in advance with a reactionbuffer and then concentrated 2-fold for adjustment, was added to theabove reaction solution, followed by further 1 hour of reaction. As aresult of the reaction, the glycoproteins formed complexes with magneticbeads via the biotinylated antibody. The complexes were adsorbed tomagnet for recovery of magnetic beads and then the solution wasdiscarded. The thus recovered complexes were washed 3 times with 500 μLof a reaction buffer and then suspended in 10 μL, of an elution buffer(0.2% SDS-containing TBS). The suspension solution was heated at 95° C.for 5 minutes, so as to dissociate and elute the glycoproteins frommagnetic beads. The thus obtained solution was designated an eluate. Atthis time, heat-denatured biotin antibodies are also mixed thereinto.Hence, 10 μL (corresponding to 20 μl of the original beads solution) ofa magnetic beads solution that had been concentrated 2-fold foradjustment by the above-mentioned technique was added to the eluate,followed by 1 hour of reaction. Thus, adsorption removal of biotinylatedantibodies was performed. The thus obtained solution was designated as aserum-derived glycoprotein solution and used for the followingexperiments.

2. Antibody-Overlay Lectin Array

An appropriate amount of the above-obtained glycoprotein solution wasadjusted with a lectin array reaction buffer (1% Triton X-100-containingphosphate-buffered saline (PBSTx)) to 60 μL. The solution was added toeach reaction vessel (8 reaction vessels were formed per glass slide)for a lectin microarray, followed by 10 or more hours of reaction at 20°C. The lectin microarray base comprising 8 reaction vessels was preparedaccording to the techniques of Uchiyama et al., (Proteomics 8, 3042-3050(2008)). Thus, a binding reaction between sugar chains on theglycoproteins and 43 types of lectin immobilized on the array substratereached an equilibrium state. Subsequently, to prevent sugar chains onantibodies for detection from binding to unreacted lectins on the base,so as to generate noise, 2 μL of a human serum-derived IgG solution(Sigma) was added and then reaction was performed for 30 minutes. Eachreaction vessel was washed 3 times with 60 μL of PBSTx and then 2 μL ofa human serum-derived IgG solution was added again. After slightstirring, a 100 ng equivalent of an antibody (biotinylated antibody fordetection) against each glycoprotein to be detected, was added to thesolution, followed by 1 hour of reaction at 20° C. Afterantigen-antibody reaction, each reaction vessel was washed 3 times with60 μL of PBSTx, and then a PBSTx solution containing a 200 ng equivalentof Cy3-labeled streptavidin was added, followed by further 30 minutes ofreaction at 20° C. After reaction, each reaction vessel was washed 3times with 60 μL of PBSTx, and then array scanning was performed usingan array scanner GlycoStation (MORITEX Corporation).

Of the thus obtained results, a typical example of each clinicalcondition resulting from the use of AGP is shown in FIG. 8 and the sameresulting from the use of Mac2BP is shown in FIG. 9. FIG. 8 shows theresults of conducting differential glycan analysis for α1 acidglycoprotein (AGP) that is one of marker glycoproteins as indices forclinical conditions of liver disease by antibody-overlay lectinmicroarray. Arrangement of lectins (array format) on the lectinmicroarray is shown on the left in the upper portion of FIG. 8. Lectinsfor which significant signals were obtained by this experiment areindicated by boldface. Signals were obtained for 15 types of lectin.Typical scan images of AGP derived from sera of hepatic cell carcinoma,cirrhosis, and chronic hepatitis patients, and healthy subjects areshown on the right in the upper portion of FIG. 8. Numerical conversionof each signal was performed using array analysis software from scandata. The results for 15 types of lectin represented by graphs are shownin the lower portion of FIG. 8. It is clearly understood thatdifferences in signal pattern arose between hepatic cell carcinoma andcirrhosis groups and hepatitis and healthy subject groups. FIG. 9 showsthe results of conducting differential glycan analysis for 90K/Mac-2Binding Protein (M2BP) that is one of marker glycoproteins as indicesfor clinical conditions of liver disease by antibody-overlay lectinmicroarray. Lectin arrangement on the lectin microarray is shown on theleft in the upper portion of FIG. 9. Lectins for which significantsignals were obtained by this experiment are indicated by boldface.Signals were obtained for 17 types of lectin. Typical scan images ofM2BP derived from sera of hepatic cell carcinoma, cirrhosis, and chronichepatitis patients, and healthy subjects are shown on the right in theupper portion of FIG. 9. Numerical conversion of each signal wasperformed using array analysis software from scan data. The results for17 types of lectin represented by graphs are shown in lower portion ofFIG. 9. It is understood that changes (increases or decreases) in signalintensity occurred depending on severity of clinical conditions.

Example 2 Identification of the Progress of Fiber Formation in the Liverby Antibody-Overlay Lectin Array Analysis of AGP Glycan-MarkerGlycoprotein as an Index for Clinical Conditions of Liver Disease

As shown in Example 1, glycoproteins were chosen by statistical analysisfrom the lectin signal information obtained by the antibody-overlaylectin array analysis of glycoproteins. The use of the thus selectedoptimum glycoprotein leads to the possibility of detecting liver diseasewith each type of clinical condition.

Accordingly, an experiment was conducted with the following proceduresusing AGP as a target molecule.

1. Narrowing Down the Number of Lectins for Distinguishing BetweenCirrhosis and Hepatitis

To narrow down the number of lectin groups exhibiting signalfluctuations with the progress of fiber formation, firstlyantibody-overlay lectin array analysis was conducted for AGP using seraof clinically diagnosed HCC, LC, and CH patients (10 cases each). Toperform more objective narrowing down, Student T test was performed forHCC-LC and LC-CH. Lectins with a risk of 0.1% or less were designated asuseful lectins. The results are shown in Table 3. As understood from theprevious experimental results (FIG. 8), as a result of the AGP arrayanalysis, signals were obtained for 15 types of lectin, and 6 types oflectin (LEL, AOL, AAL, MAL, STL, and PHAE) out of the 15 types exhibitedsignificant differences (risk of 0.1% or less as a Student T testresult). In this experiment, lectin DSA exhibited the fewest signalfluctuations, but it also exhibited high reproducibility. Regarding thelectin DSA, we found effectiveness in the normalization of the thusobtained data and determined that after scanning, all data subjected tonumerical conversion had been normalized with DSA signals.

TABLE 3 CV (average) After DSA Net intensity nomalization (0.53) (0.49)T-test (P) T-test (P) Lectin HCC-CH LC-CH HCC-CH LC-CH LEL 2.60E−107.30E−06 3.20E−16 1.70E−08 AOL 4.70E−08 7.40E−09 1.50E−07 1.10E−07 AAL6.50E−08 1.20E−07 1.00E−07 1.20E−07 MAL 1.10E−07 1.60E−04 1.30E−071.70E−04 STL 4.30E−07 2.00E−04 5.30E−11 3.10E−07 PHAE 1.20E−04 8.80E−052.90E−05 2.40E−05 ABA 5.60E−01 2.00E−02 5.60E−01 2.40E−03 PHAL 1.90E−034.40E−02 1.40E−03 2.30E−02 SSA 2.80E−02 2.00E−02 3.50E−04 1.50E−06RCA120 1.70E−01 3.20E−02 7.60E−02 3.70E−04 WGA 5.20E−02 2.50E−015.20E−03 3.50E−02 SNA 1.60E−01 1.30E−01 4.60E−02 3.30E−03 UDA 4.40E−018.70E−01 5.00E−01 6.80E−01 DSA 9.70E−01 7.10E−01 — — TJAI 9.10E−018.50E−01 9.30E−01 6.70E−01

2. Narrowing Down of the Number of Lectins for Identifying the Progressof Fiber Formation

Next, antibody-overlay lectin microarray was performed for 125 cases ofa group of patients infected by hepatitis virus and pathologicallydiagnosed by liver biopsy for staging of fiber formation according tothe procedures of Example 1. In addition, the results of staging forfiber formation in the liver in 125 cases are as follows: F0 and F1 (33cases), F2 (32 cases), F3 (31 cases), and F4 (29 cases). According tothe above procedures, lectins statistically useful for identificationwere narrowed down. As a result, the results for top 6 types of lectinare shown in Table 4. The thus obtained 6 lectins ranked high andparticularly LEL, AOL, and MAL were selected as the most effectivelectins for identification. Hence, detailed data analysis was conductedfor the 3 types of lectin.

TABLE 4 N = 125 T-test (P) Lectin F0-3 vs F4 F3 vs F4 LEL 1.74E−162.53E−08 AOL 8.37E−16 5.30E−05 AAL 1.98E−13 3.46E−04 MAL 2.83E−148.05E−06 STL 1.41E−06 5.79E−04 PHAE 1.18E−12 2.36E−04

FIG. 10 shows the correlations between the progress of fiber formationin the liver and changes in lectin signal intensity obtained byantibody-overlay lectin array analysis of AGP. Each signal wasnormalized with the signal of DSA lectin and a numerical value isexpressed as relative signal intensity when the DSA signals aredesignated as 100%. The results shown in A in the upper portion of FIG.10 were obtained by performing lectin array analysis for 125 cases forwhich staging (F) of fiber formation had been performed by pathologicalanalysis after liver biopsy. The distribution of the lectin signal ateach stage is shown with a box-whisker plot. The upper end and the lowerend of each box indicate a point of 75% and a point of 25%,respectively. The upper end and the lower end of each whisker indicate apoint of 95% and a point of 5%, respectively. A transverse line in eachbox indicates the median value and “x” indicates the average value.Student-T test was performed to test significant differences betweencirrhosis (F4) and chronic hepatitis (at stage F0, 1, 2, or 3). Eachresult with a risk of P<0.0001 is indicated with *. Also as a control,the distribution of numerical values for blood platelets, which is usedas an index for fiber formation in the liver upon general biochemicalexamination. As a result, the intensity of AOL signal increased with theprogress of fiber formation. It was demonstrated that chronic hepatitis(F0-3) and cirrhosis (F4) can be sufficiently distinguished from eachother based on differences in intensity. On the other hand, it wasrevealed that the intensities of MAL and LEL signals decreased with theprogress of fiber formation.

The results of measuring with time AOL, MAL, and LEL signal fluctuationsin the same patient are shown in B in the lower portion of FIG. 10.Antibody-overlay lectin microarray analysis was conducted for AGP in aseries of specimens (serum samples collected at different times) from asingle case of a cirrhosis patient or a hepatic cell carcinoma patient,and then the relative signal values of AOL, MAL, and LEL afternormalization with DSA signals were plotted in B in the lower portion ofFIG. 10. The time axis was set so that the date of the definitivediagnosis of cirrhosis and hepatic cell carcinoma was designated as “day0.” The intensity of AOL signal increased with time and that of MALsignal decreased with time, leading to the reflection of the progress offiber formation in the liver. On the other hand, the intensity of LELsignals or the number of blood platelets, which is used as a simplefiber formation marker, exhibited rapid fluctuations at certain times,and did not clearly express the progress of fiber formation.

It was demonstrated by the above results that the independent use of ora combined use of AOL and MAL signal fluctuations makes it possible toidentify the progress of fiber formation in the liver.

Example 3 Detection of Cirrhosis by Antibody-Overlay Lectin ArrayAnalysis for AGP Glycan-Marker Glycoprotein as an Index for ClinicalConditions of Liver Disease

It was considered based on the results of Example 2 that determinationof a cut-off value of each lectin (signals) or a combination of lectinsignals on the basis of the progress of fiber formation in the liverenables detection of cirrhosis. Hence, an experiment was conducted withthe following procedures.

1. Determination of Lectin Signal.Cut-Off Values for Detection ofCirrhosis

First, a cut-off value was determined for each lectin in order to detecta patient with cirrhosis from among patients infected by hepatitisvirus. For this purpose, a receiver operating characteristic curve (ROCcurve) for distinguishing F4 (cirrhosis) from the other stages (F1-F3)was created for 80 cases of pathologically diagnosed patients (F1, F2,F3, and F4 (20 cases each)) with the use of data obtained bynormalization of 2 types of lectin signal narrowed down in Example 2with DSA lectin signals. The results are shown on the left in FIG. 11.FIG. 11 shows, in addition to a curve of a case in which AOL signal orMAL signal was independently used, a curve representing numerical valuesobtained by an equation (relative signal intensity of AOL)×1.5−(relativesignal intensity of MAL), in which 2 signals were used. AUC (area undercurve) values representing the area of the lower part under curve werefound, so as to assess the diagnostic ability of each technique. Also, apoint on a curve, which is located closest to the point of 100%sensitivity and the point of 100% specificity, in other words, a contactpoint between a line parallel to the line of Y=X and the ROC curve, wasdesignated as “optimum specificity and sensitivity” and in the peripherycut-off values were determined. These numerical values are shown in theright table in FIG. 11. With the use of the thus obtained cut-offvalues, a blind test was performed for 45 cases and 43 cases of patientsdefinitively diagnosed as having hepatitis and cirrhosis by pathologicaldiagnosis or diagnostic imaging.clinical diagnosis. The results areshown in the column of “Validation set” in the right table in FIG. 11.Cirrhosis was determined to be positive and hepatitis was determined tobe negative, and the detected number thereof is listed in the table. Onthe basis of the ROC curve, each cut-off value was determined at a pointwhere the best sensitivity and the best specificity were exhibited. Thecut-off values were 8% in the case of AOL and 11.8% in the case of MAL.Also, in the combination system, the fewest number of false-negativepatients and the fewest number of false-positive patients were confirmedand the accuracy (%) ((total number of patients−(the number offalse-positive and false-negative patients))/(total number ofpatients)×100) was high.

2. Detection of Cirrhosis

Antibody-overlay lectin array was performed for 45 cases of clinicallydiagnosed chronic hepatitis patients and 43 cases of clinicallydiagnosed cirrhosis patients according to the procedures in Example 2using AGP as a target molecule. All signals were normalized with thesignal of DSA lectin, which had been designated as 100%. The numericalvalues were determined to be positive or negative using theabove-mentioned cut-off values, so that cirrhosis detection was tested.As a result, when AOL signals were used, the detection ability wasrepresented by the sensitivity of 86.1%, the specificity of 91.1%, andthe accuracy of 88.6%. When MAL signals were used, the detection abilitywas represented by the sensitivity of 90.7%, the specificity of 88.9%,and the accuracy of 89.8%. Significantly high-level detection waspossible in both cases, such that the accuracy exceeded 85%. Moreover,the detection ability was examined using an equation with a combined useof two signals ((relative signal intensity of AOL)×1.5−(relative signalintensity of MAL)). Thus, the most reliable cirrhosis detection resultwas obtained, such that the sensitivity was 95.4%, the specificity was91.1%, and the accuracy was 93.2%. It is particularly worth noting thatthe detection ability was overwhelmingly higher than those of knowntechniques for detection of disease-specific glyco-alterations in AGPusing AAL lectin or RCA lectin. These results agree with the result ofthe narrowing-down step using antibody-overlay lectin array in which AALand RCA120 were inferior to AOL and MAL (see Table 3 and Table 4).

Example 4 Determination of Therapeutic Effects of an Interferon Using aMarker Capable of Monitoring the Progress of Fiber Formation in theLiver

It was demonstrated based on the results of Example 2 that the progressof fiber formation can be monitored through observation of AOL and MALsignal fluctuations. Hence, the therapeutic effects of an interferonthat is an antiviral agent were experimentally determined using AOL/DSAor MAL/DSA as a parameter indicating the progress of fiber formation.The following experiment was conducted for type C hepatitis patients ofa sustained virological responder (SVR) group for which the therapeuticeffects of the interferon had been confirmed and a non-virologicalresponder (NVR) group for which no therapeutic effect thereof had beenconfirmed. After interferon treatment, patients' sera from bloodcollected with time were subjected to enrichment of AGP in scrum andantibody-overlay lectin microarray analysis by techniques similar tothose in Example 1. After normalization with DSA signals, relativesignal values of AOL and MAL were calculated. The time course changes ineach signal (typical results of the SVR group and the NVR group (2 caseseach)) are shown in FIG. 12. The time axis was set so that the date ofblood collection immediately after treatment was designated as “day 0.”Regarding relative binding signals, the relative value immediately aftertreatment was designated as “0.” In the SVR cases, MAL signals increasedwith time, but AOL signals decreased or no AOL signal was detected.Specifically, in these cases, fiber formation tended to be alleviated.On the other hand, in NVR cases, AOL signals increased with time and MALsignals remained almost unchanged. Specifically, in these cases, atendency was observed such that fiber formation was not alleviated butrather worsened (e.g., fiber formation progressed). As described above,it was revealed that the effects after interferon treatment can bedetermined by a blood test.

Example 5 1. 1St Rapid Measurement Method (Manual Method) 1-1.Preparation of Reagent

Preparation of R1 reagent: R1 reagent 1 was prepared by adding 5 μg/mLbiotinylated DSA (J-OIL MILLS, INC.) to buffer A (PBS-1% TritonX,pH7.4). R1 reagent 2 was prepared by adding 5 μg/mL biotinylated MAL(Vector) to buffer A. R1 reagent 3 was prepared by adding 2.5 μg/mLbiotinylated AOL to buffer A. In addition, biotinylated AOL used hereinwas prepared by biotinylation of AOL (Tokyo Kasei Kogyo Co., Ltd.) usinga biotin labeling kit (DOJIDO).

Preparation of R2 reagent: An R2 reagent was prepared by adding magneticparticles (number average particle size: 2 μm) to which streptavidin hadbeen immobilized to buffer A to 0.5 w/v %.

Preparation of R3 reagent: A solution containing a 0.025 U/mLALP-labeled mouse anti-AGP monoclonal antibody, 0.1 M MES(2-(N-Morpholino)ethanesulfonic acid, pH 6.5), 0.15 M sodium chloride, 1mM magnesium chloride, 0.1 mM zinc chloride, 0.1 w/v % NaN₃, and 0.5 w/v% casein Na was prepared and then designated as R3 reagent 1. R3 reagent2 was prepared in a manner similar to that for R3 reagent 1 except forthe use of a 0.5 U/mL ALP-labeled mouse anti-AGP monoclonal antibodyinstead of a 0.025 U/mL ALP-labeled mouse anti-AGP monoclonal antibody.

Preparation of R4 reagent: A solution containing 0.1 M2-amino-2-methyl-1-propanol (AMP, pH 9.6), 1 mM magnesium chloride, and0.1 w/v % NaN₃ was prepared and then designated as an R4 reagent.

Preparation of R5 reagent: CDP-Star with Sapphirine-II (luminescentsubstrate for ALP, Applied Biosystems) was designated as an R5 reagent.

Preparation of washing reagent: A solution containing 20 mM tris(pH7.4), 0.1 w/v % Tween20, 0.1 w/v % NaN₃, and 0.8 w/v % sodiumchloride was prepared, and then designated as a washing reagent.

1-2. Confirmation of Dilution Linearity of DSA

AGP in Consera (normal human serum, Nissui Pharmaceutical Co., Ltd.) wasrecovered in buffer B (TBS-0.5% TritonX-0.1% SDS) using an anti-AGPantibody in a manner similar to that for enrichment in Example 1 andthen used as a sample. The thus recovered sample was diluted 1-fold,½-fold, ¼-fold, ⅛-fold, and 1/16-fold, respectively, with buffer B sothat diluted samples were prepared.

R1 reagent 1 (110 μL) was added to 30 μL of each diluted sample. After 2minutes of reaction at room temperature, 30 μL of the R2 reagent wasadded and then reaction was performed for 30 minutes at roomtemperature. Magnetic particles bearing the complex of DSA and AGP werecollected for B/F separation, the separated magnetic particles werewashed using a washing reagent, and then the solution was discarded.This treatment was performed 4 times. R3 reagent 1 (100 μL) was added tothe washed magnetic particles, followed by 20 minutes of reaction atroom temperature, so that AGP in the complex on magnetic particles wasreacted with the ALP-labeled mouse anti-AGP monoclonal antibody.Collected magnetic particles bearing the complex of the ALP-labeledmouse anti-AGP monoclonal antibody, AGP, and DSA were subjected to theremoval of liquid components (B/F separation), the thus separatedmagnetic particles were washed with a washing reagent, and then thesolution was discarded. This treatment was performed 4 times.Complex-bearing magnetic particles were dispersed in 50 μL of the R4reagent, 100 μl, of the R5 reagent was added, and then chemiluminescenceintensity due to ALP was measured as a photo count value using aluminescence measurement apparatus. The results are shown in FIG. 13. Asshown in FIG. 13, good linearity, R²=0.99, was exhibited in the DSAmeasurement system.

1-3. Confirmation of Dilution Linearity of MAL

An experiment was conducted for confirmation of dilution linearity in aMAL measurement system in a manner similar to that in “confirmation ofdilution linearity of DSA” except for the use of R1 reagent 2 instead ofR1 reagent 1 and the use of R3 reagent 2 instead of R3 reagent 1. Theresults are shown in FIG. 14. As shown in FIG. 14, good linearity,R²=0.99, was exhibited in the MAL measurement system.

1-4. Confirmation of Dilution Linearity of AOL

An experiment was conducted for confirmation of dilution linearity in anAOL measurement system in a manner similar to that in “confirmation ofdilution linearity of DSA” except for the use of HCV-positive bloodplasma 2 (Millenium Biotech) instead of Consera and the use of R1reagent 3 instead of R¹ reagent 1. The results are shown in FIG. 3. Asshown in FIG. 15, good linearity, R²=0.98, was exhibited in the AOLmeasurement system.

1-5. Measurement of DSA, MAL, and AOL for Various Commercially AvailableSpecimens

Consera (normal human serum, Nissui Pharmaceutical Co., Ltd.), normalhuman serum (TRINA), and HCV-positive blood plasma 1 and 2 (MilleniumBiotech) were each subjected to AGP separation using an anti-AGPantibody in a manner similar to that for enrichment in Example 1. Theresultants were then each recovered in a buffer (TBS-0.5% TritonX-0.1%SDS) and then used as samples for measurement. Also, a buffer alone wasused as a sample (NC) for blank measurement.

R1 reagent 1 (110 μL) was added to 30 μL of each measurement sample.After 2 minutes of reaction at room temperature, 30 μL of the R2 reagentwas added and then reaction was performed at room temperature for 30minutes. Magnetic particles bearing the complex of DSA and AGP werecollected for B/F separation, the separated magnetic particles werewashed with a washing reagent, and then the solution was discarded. Thistreatment was performed 4 times. R3 reagent 1 (100 μL) was added to thewashed magnetic particles, reaction was performed at room temperaturefor 20 minutes, and then AGP in the complex on magnetic particles wasreacted with the ALP-labeled mouse anti-AGP monoclonal antibody.Magnetic particles bearing the complex of the ALP-labeled mouse anti-AGPmonoclonal antibody, AGP, and DSA were collected for B/F separation, theseparated magnetic particles were washed with a washing reagent, andthen the solution was discarded. This treatment was performed 4 times.Complex-bearing magnetic particles were dispersed in 50 μL of the R4reagent and then 100 μL of the R5 reagent was added. Chemiluminescenceintensity in the DSA measurement system was measured as a photo countvalue using a luminescence measurement apparatus. The time required formeasurement was 65 minutes. Measurement results are shown in Table 5.

Also, chemiluminescence in the MAL measurement system was measured in amanner similar to that in the DSA measurement system except for the useof R1 reagent 2 instead of R1 reagent 1 and the use of R3 reagent 3instead of R3 reagent 1. The results are shown in Table 5. Also,chemiluminescence in the AOL measurement system was measured in a mannersimilar to that in the DSA measurement system except for the use of R1reagent 3 instead of R1 reagent 1. The results are shown in Table 5.Also, measurement results obtained using MAL and AOL are normalized withthe measurement result obtained using DSA and then the thus obtainedvalues are shown in Table 5, in addition to FIG. 16 and FIG. 17.

TABLE 5 DSA MAL AOL MAL/DSA AOL/DSA NC 134464 4591 39520 Consera 9470412566153 122283 315.24 10.19 Normal 920232 2057193 145020 261.22 13.43human serum HCV-1 1154871 3063570 181898 299.78 13.95 HCV-2 8482901093355 404969 152.53 51.20

1-6. Measurement by a Lectin Array Method for Various CommerciallyAvailable Specimens

Each measurement sample in the above 1-5 was subjected to measurement byan antibody overlay method using lectin arrays used in Example 1. Thetime for measurement required by the lectin arrays method was about 18hours. The results are shown in Table 6, in addition to FIG. 18 and FIG.19.

TABLE 6 Measurement results by lectin array MAL/DSA AOL/DSA Consera 15.42.8 Normal human serum 11 4.7 HCV-1 15.2 4 HCV-2 5.4 15.4

Regarding measurement results for MAL subjected to normalization withDSA, it was demonstrated that the measurement results obtained by the1^(st) measurement method shown in the embodiment (shown in FIG. 16)exhibited a good correlation with the measurement results obtained bythe lectin array method shown in FIG. 18. Also, regarding themeasurement results for AOL subjected to normalization with DSA, themeasurement results obtained by the 1^(st) rapid measurement methodshown in FIG. 17 exhibited a pattern similar to that of and thus a goodcorrelation with the measurement results obtained by the lectin arraymethod shown in FIG. 19.

2. The 2nd Rapid Measurement Method (Automatic Measurement Method) 2-1.Preparation of Reagent

Preparation of R1 reagent: Buffer A (PBS-1% TritonX, pH7.4) wasdesignated as an R1 reagent.

Preparation of R2 reagent: Magnetic particles (number average particlesize: 2 μm) to which streptavidin had been immobilized was added tobuffer A to 0.5 w/v % and then 2.5 μg/mL biotinylated DSA (J-OIL MILLS,INC.) was added, followed by 30 minutes of stirring at room temperature.After stirring, the magnetic particles were collected and thenprecipitated, so that solution components were discarded. Buffer A wasadded to the resultant and then stirred. Magnetic particles werecollected and then precipitated, so that solution components werediscarded. This procedure was repeated 3 times. Buffer A was added tothe resultant so that the concentration of magnetic particles was 0.5w/v %. The thus obtained solution containing DSA-bearing magneticparticles was designated as R2 reagent 1. R2 reagent 2 containingMAL-bearing magnetic particles was prepared in a manner similar to thatfor R2 reagent 1 except for the use of 25 μg/mL biotinylated MAL(Vector) instead of 2.5 μg/mL biotinylated DSA (J-OIL MILLS, INC.). R2reagent 3 containing AOL-bearing magnetic particles was prepared in amanner similar to that for R2 reagent 1 except for the use of 25 μg/mLbiotinylated AOL instead of 2.5 μg/mL biotinylated DSA (J-OIL MILLS,INC). In addition, biotinylated AOL used herein was prepared bybiotinylation of AOL (Tokyo Kasei Kogyo Co., Ltd.) using abiotin-labeling kit (DOJIDO).

Preparation of R3 reagent: A solution containing 0.1 U/mL ALP(Recombinant AP, EIA Drade, CR 03535452)-labeled mouse anti-AGPmonoclonal antibody-Fab′, 0.1 M MES (2-(N-Morpholino) ethanesulfonicacid, pH6.5), 0.15 M sodium chloride, 1 mM magnesium chloride, 0.1 mMzinc chloride, 0.1 w/v % NaN₃, and 0.25 w/v % casein Na was prepared anddesignated as R3 reagent 1. R3 reagent 2 was prepared in a mannersimilar to that for R3 reagent 1 except for the use of 0.1 w/v % BSAinstead of 0.25 w/v % casein Na.

In addition, in the above 1st rapid measurement method (manual method),ALP that had not been deglycosylated was used, but in the 2nd rapidmeasurement method, deglycosylated ALP was used.

Preparation of R4 reagent: A solution containing 0.1 M2-amino-2-methyl-1-propanol (AMP, pH9.6), 1 mM magnesium chloride, and0.1 w/v % NaN₃ was prepared and designated as an R4 reagent.

Preparation of R5 reagent: CDP-Star with Sapphirine-II (luminescentsubstrate for ALP, Applied Biosystems) was designated as an R5 reagent.

Preparation of washing reagent: A solution containing 20 mM tris(pH7.4), 0.1 w/v % Tween20, 0.1 w/v % NaN₃, and 0.8 w/v % sodiumchloride was prepared and designated as a washing reagent.

2-2. Confirmation of Dilution Linearity of a Measurement System UsingDSA

AGP in Consera (normal human serum, Nissui Pharmaceutical Co., Ltd.) wasrecovered in buffer B (TBS-0.5% TritonX-0.1% SDS) using an anti-AGPantibody in a manner similar to that for enrichment in Example 1 andthen used as a sample. The thus recovered sample was diluted 1-fold,½-fold, ¼-fold, ⅛-fold, and 1/16-fold, respectively, with buffer B sothat diluted samples were prepared.

Conditions for the operation of a full-automatic immunoassay systemHISCL2000i (Sysmex) were changed to the following conditions.Chemiluminescence (photo count value) was measured for each dilutedsample using the system.

Each diluted sample (30 μl) was dispensed into a vessel. After 2.25minutes of incubation at 42° C., 30 μL of the R2 reagent 1 wasdispensed, followed by 2.5 minutes of reaction at 42° C. Furthermore,100 μL of the R³ reagent 1 was dispensed and then a reaction wasperformed at 42° C. for 2.75 minutes. Magnetic particles were collectedby magnetic separation, so that the solution was vacuumed and discarded.The washing reagent was dispensed, magnetic particles were dispersed inand washed with the washing reagent, magnetic particles were collectedby magnetic separation, and thus the solution was vacuumed anddiscarded. This treatment was repeated 3 times. The R⁴ reagent (50 μL)was dispensed, 100 μL of the R⁵ reagent was dispensed, and thenchemiluminescence was measured. The above measurement was performed 3times and then the results are shown in FIG. 20. As shown in FIG. 20,good linearity, R²=0.98, was exhibited in the DSA measurement system.

2-3. Confirmation of Dilution Linearity of a Measurement System UsingMAL

An experiment was conducted for confirmation of dilution linearity inthe MAL measurement system in a manner similar to that in “Confirmationof dilution linearity of a measurement system using DSA” except for theuse of R2 reagent 2 instead of R2 reagent 1 and the use of R3 reagent 2instead of R3 reagent 1. The results are shown in FIG. 21. As shown inFIG. 21, good linearity, R²=0.99, was exhibited in the MAL measurementsystem.

2-4. Confirmation of Dilution Linearity of a Measurement System UsingAOL

An experiment was conducted for confirmation of dilution linearity inthe AOL measurement system in a manner similar to that in “Confirmationof dilution linearity of DSA” except for the use of HCV-positive bloodplasma 2 (Millenium Biotech) instead of Consera and the use of R2reagent 3 instead of R2 reagent 1. The results are shown in FIG. 22. Asshown in FIG. 22, good linearity, R²=0.99, was exhibited in the AOLmeasurement system.

2-5. Measurement Using DSA, MAL, and AOL for Various CommerciallyAvailable Specimens

Consera (normal human serum, Nissui Pharmaceutical Co., Ltd.), normalhuman serum (TRINA), and HCV-positive blood plasma 1 and 2 (MilleniumBiotech) were each subjected to separation of AGP by animmunoprecipitation method using an anti-AGP antibody. Resultants wereeach recovered in buffer B (TBS-0.5% TritonX-0.1% SDS) and used assamples for measurement. Also, buffer B alone was used as a sample forblank measurement.

Each measurement sample was measured using a full-automatic immunoassaysystem HISCL2000i under conditions similar to those for “confirmation ofdilution linearity of a measurement system using DSA,” “confirmation ofdilution linearity of a measurement system using MAL,” and “confirmationof dilution linearity of a measurement system using AOL.” The timerequired for each measurement was 17 minutes. The results are shown inTable 7, FIG. 23, and FIG. 24.

TABLE 7 DSA MAL AOL KAL/DSA AOL/DSA Consera 13757054 1377260 132648 10.01.0 Normal 17770250 1549266 417612.7 8.7 2.4 human serum HCV-1 198851061877027 185538 9.4 0.9 HCV-2 15225585 825426.3 1776264 5.4 11.7

Regarding the measurement results obtained using MAL subjected tonormalization with the measurement result obtained using DSA, it wasdemonstrated that the measurement results obtained by the 2^(nd) rapidmeasurement method shown in FIG. 23 exhibited a good correlation withthe measurement results obtained by the lectin array method shown inFIG. 18. Also, regarding the measurement results obtained using AOLsubjected to normalization with measurement results obtained using DSA,the measurement results obtained by the 2nd rapid measurement methodshown in FIG. 24 exhibited a pattern similar to that of and thus a goodcorrelation with the measurement results obtained by the lectin arraymethod shown in FIG. 19.

2-6. Measurement of Clinical Specimens Using DSA, MAL, and AOL

Serum 1 and serum 2 from patients at fiber formation stage F1, serum 3and serum 4 from patients at fiber formation stage F2, serum 5 and serum6 from patients at fiber formation stage F3, and serum 7 and serum 8from patients at fiber formation stage F4 were each subjected toseparation of AGP using an anti-AGP antibody in a manner similar to thatfor enrichment in Example 1, and then recovered in buffer B (TBS-0.5%TritonX-0.1% SDS). The resultants were used as samples for measurement.Also, buffer B alone was used as a sample for blank measurement.

Each measurement sample was measured using a full-automatic immunoassaysystem HISCL2000i under conditions similar to those for “confirmation ofdilution linearity of a measurement system using DSA,” “confirmation ofdilution linearity of a measurement system using MAL,” and “confirmationof dilution linearity of AOL.” The time required for each measurementwas 17 minutes. The results are shown in Table 8, FIG. 25, and FIG. 26.

TABLE 8 Specimen 1 2 3 4 5 6 7 8 MAL/DSA 12.8 11.5 11.5 10.7 9.4 8.7 5.51.1 AOL/DSA 0.3 0.7 0.7 1.2 3.2 3.9 20.2 117.0

2-7. Measurement of Clinical Specimens by a Lectin Array Method

Each measurement sample of the above 2-6 was measured by an antibodyoverlay method using lectin arrays used in Example 1. The time requiredfor measurement by the lectin array method was about 18 hours. Theresults are shown in Table 9, FIG. 27, and FIG. 28.

TABLE 9 Specimen 1 2 3 4 5 6 7 8 MAL/DSA 27.3 24.3 25.6 24.0 18.1 18.98.5 1.3 AOL/DSA 0.5 3.0 4.0 5.0 11.8 13.1 37.1 73.6

It was demonstrated based on the results shown in FIG. 25 to FIG. 28that, also in the case of clinical specimens, the 2^(nd) rapidmeasurement method exhibited a pattern similar to that of and thus agood correlation with the lectin array method.

Example 6 Validation of the 2^(nd) Rapid Measurement Method 1. AnalysisUsing 125 Cases of HCV-Infected Patients

It was revealed by Example 5 that the 2^(nd) rapid measurement methodexhibited a pattern similar to that of a lectin array. To demonstratethe fact in more cases, the 2^(nd) rapid measurement method wasperformed according to the procedures 2-6 in Example 5 for 125 cases ofthe patient group (same as that in Example 2) infected with hepatitisvirus and pathologically diagnosed by liver biopsy for staging for fiberformation. In addition, the results of staging for fiber formation inthe liver performed for 125 cases were: 33 cases at stages F0 and F1, 32cases at stage F2, and 31 cases at F3, and 29 cases at F4.

FIG. 29 shows correlations between the progress of fiber formation inthe liver and changes in lectin signal intensity obtained by the 2ndrapid measurement method performed for AGP. Signals on each lectin werenormalized with signals on DSA lectin, numerical values were expressedas relative signal intensities when DSA signals (signals on DSA) weredesignated as 100%. On the left in FIG. 29, the distribution of lectinsignals at each stage is shown with box-whisker plots when the 2^(nd)rapid measurement method was performed for 125 cases for which staging(F) for fiber formation had been performed by pathologic(al) analysisafter liver biopsy. MAL/DSA values were each found from the 3 types oflectin value obtained by the 2^(nd) rapid measurement method and thenbox-whisker plots were created as shown in the upper left portion.AOL/DSA values were also found and then box-whisker plots were created,as shown in the lower left portion. The upper end and the lower end ofeach box indicate a point of 75% and a point of 25%, respectively. Theupper end and the lower end of each whisker indicate a point of 95% anda point of 5%, respectively. A transverse line in each box indicates themedian value and “x” indicates the average value. All results stronglyresembled to the results of the lectin array. The signal intensity ofAOL increased with the progress of fiber formation. It was demonstratedthat chronic hepatitis (F0-3) and cirrhosis (F4) can be sufficientlydistinguished from each other based on a difference in intensity. On theother hand, the signal intensity of MAL was found to decrease with theprogress of fiber formation. To support the fact that a correlation waspresent between the results of the lectin array and the results of the2^(nd) rapid measurement method, numerical values obtained from the twowere plotted two-dimensionally. The graph is shown on the right in FIG.29. It was revealed from the graph that there is a strong correlationbetween the results of the lectin array and the results of the 2^(nd)rapid measurement method.

2. Detection of Cirrhosis

Detection of cirrhosis by the 2^(nd) rapid measurement method wasattempted by procedures similar to those for detection of cirrhosisusing lectin arrays described in Example 3. An ROC curve fordistinguishing F4 (cirrhosis) from the other (F1-F3) was created for 125cases of the above pathologically diagnosed patients using data obtainedby normalization of signals on 2 types of lectin with the signals on DSAlectin. Also, a point on a curve, which is located closest to the pointof 100% sensitivity and the point of 100% specificity, in other words, acontact point between a line parallel to the line of Y=X and the ROCcurve, was designated as “optimum specificity and sensitivity” and inthe periphery cut-off values were determined. A combination equation(AOL/DSA×1.8−MAL/DSA) optimum for detection of cirrhosis was found basedon the thus obtained cut-off value for each lectin. Accordingly, a blindtest was performed for 45 cases and 43 cases of patients definitivelydiagnosed as having hepatitis and cirrhosis by pathological diagnosis ordiagnostic imaging.clinical diagnosis.

As a result, the detection ability examined using the combinationequation (AOL/DSA×1.8−MAL/DSA) was represented by the sensitivity of88.3%, the specificity of 91.1%, and the accuracy of 89.8%.

Next, to examine the frequency of false-positive results from theequation, sera from 100 healthy subjects were also analyzed by the2^(nd) rapid measurement method. Determination of cirrhosis wasperformed using the equation. As shown in FIG. 30, false-positive ratewas found to be 5%, such that the results of lectin arrays wereconsistent with those of the false-positive specimens.

Example 7 Identification of the Progress of Fiber Formation in the Liverby Antibody-Overlay Lectin Array Analysis of a Candidate Glycan-MarkerGlycoprotein M2BP as an Index for Clinical Conditions of Liver Disease

As described in Example 2, a possibility was found also for M2BP, suchthat each type of clinical condition of liver disease can be detectedbased on the lectin signal information obtained by antibody-overlaylectin array analysis. Hence, an experiment for examination of acorrelation with fiber formation in the liver was conducted.Antibody-overlay lectin microarray was performed according to theprocedures in Example 2 for 125 cases of a group of patients (same asthose in the experiment for AGP) infected with hepatitis virus andpathologically diagnosed by liver biopsy for staging for fiberformation. Graphs showing correlations between 6 lectins (out of 17types of lectin for which binding signals had been generated), for whichsignal changes had been confirmed by a significance difference test withthe progress of fiber formation, and the progress of fiber formationwere created, as shown in FIG. 31. The distribution of the lectinsignals at each stage is shown with a box-whisker plot. The upper endand the lower end of each box indicate a point of 75% and a point of25%, respectively. The upper end and the lower end of each whiskerindicate a point of 95% and a point of 5%, respectively. A transverseline in each box indicates the median value and “x” indicates theaverage value. In addition, the vertical axis of each graph representsrelative values when DSA signals were designated as 100%. It wasrevealed that signal intensities of RCA120, AAL, TJAII, WFA, and BPLincreased with the progress of fiber formation, while the signalintensity of LEL decreased with the progress of fiber formation. Ofthese lectins, RCA120 and AAL are thought to be suitable in view ofmonitoring the progress of fiber formation. Also, signals were generatedon TJAII, WFA, and BPL when the cirrhosis stage reached F4. Moreover,TJAII, WFA, and BPL signals were varied significantly in the case of F4,suggesting a possible use of TJAII, WFA, and BPL as effective markersfor discriminating a high risk group for cancer.

Example 8 Discrimination of a High Risk Group for Liver Cancer byDetection of Glyco-Alterations in M2BP 1. Selection of Lectins Suitablefor Discriminating a High Risk Group for Liver Cancer

A group of lectins with signal intensities that increased with theprogress of fiber formation in the liver was found in Example 7. Todiscriminate lectins capable of discriminating a high risk group forcancer from the group of lectins, antibody-overlay lectin microarrayanalysis of M2BP was conducted for sera from 7 cases of preoperative andpostoperative patients with hepatic cell carcinoma. The experimentalprocedures were employed according to Example 1. Also, with the use of afull-automatic fluorescence immunoassay apparatus (μTAS Wako i30; WakoPure Chemical Industries, Ltd.) and a special-purpose reagent, an amountof known hepatic cell carcinoma marker AFP in the blood and benigndisorder.hepatic cell carcinoma distinguishing marker AFP-L 3% were alsomeasured. The resulting lectin signal patterns are partially shown inFIG. 32. All parameters including AFP-L 3% did not always exhibitpost-operative signal decreases in all the 7 cases. This means that allof these markers are not cancer detection markers. An object of theexperiment is to select candidate markers for discriminating a high riskgroup for hepatic cell carcinoma. With regard to this point, a desiredcandidate marker exhibits a pattern analogous to that exhibited by AFP-L3%. As predicted in Example 7, WFA and BPL lectins exhibited patternsanalogous to that of AFP-L 3%. Moreover, the signal intensity of WFA wasstronger and more stable than that of BPL, so that WFA was selected as apowerful candidate lectin.

2. Detection of WFA-Binding M2BP by a Lectin-Antibody Sandwich ELISAMethod

The best mode for detection of WFA-binding M2BP as a biomarker fordiscriminating a high risk group for liver cancer can be a method fordetecting and identifying biomarkers contained in serum by a clinicallyapplicable convenient means with clinically acceptable performance. Theresults of sandwich detection analysis using an anti-M2BP antibodyoverlay-WFA well plate (see FIG. 33) are as shown below. In addition,serum can be directly added to a WFA well plate in this technique. Thereason of this is that very few glycoprotein molecules contained inserum can bind to WFA and in patients of a high risk group for cancer,M2BP is one of molecules with the highest concentration in the blood.

(Experimental Methods)

Fifty (50) μL each of biotinylated WFA (Vector, 5 μg/mL) dissolved in aPBS buffer was added to each well of a microtiter plate (GreinerBio-One, 96-well flat-bottomed streptavidin-coated plate) and thenmaintained at room temperature for 2 hours, so that WFA was immobilizedto a support. The plate was washed twice with a wash (0.1% Tween20-containing PBS (300 μL)) to remove unbound WFA so that thepreparation of a WFA-immobilized well plate was completed.

Each sample (1 μL) was diluted with 50 μL of the above wash and then thediluted sample was added to the WFA-immobilized well plate prepared in1, followed by 2 hours of binding reaction at room temperature. Afterreaction, each well was washed 5 times with 300 μL of the above wash, sothat unbound proteins were removed. A detection agent (goat anti-M2BPpolyclonal antibody solution; R&D Systems) adjusted in advance with awash to 1.0 μg/mL was added with 50 μL per well, followed by 2 hours ofantigen-antibody reaction at room temperature.

After washing with 300 μL of a wash to remove unbound antibodies, ananti-goat IgG antibody-HRP solution (Jackson immuno Research) diluted10,000-fold in advance with a wash was added with 50 μL per well andthen the resultants were maintained at room temperature for 1 hour. Eachwell was washed 5 times with 30 μL of a wash, and then an ULTRA-TMBsolution (Thermo) as a coloring reagent was added with 100 μL per well,followed by 10 minutes of coloring reaction. A 1 M H₂SO₄ solution wasadded with 100 μL per well to stop the reaction, and then absorbance wasmeasured at 450 nm using a plate reader. The serum of a healthy subjectwas designated as a negative control (N), numerical conversion of thethus obtained signals was performed to find S/N ratios, and then theresulting data were used for the following analysis.

Results

First, assay was performed for sera from 7 cases of the abovepreoperative and postoperative hepatic cell carcinoma patients. As aresult, signal patterns that strongly resembled to the results in FIG.32 were obtained (upper portion of FIG. 34). Of these cases, 3 cases forwhich postoperative blood collection had been sequentially performedwere subjected to measurement of the amount of WFA-binding M2BP. Inaddition, patients A and B were postoperative recurrence cases, andpatient E was a no-recurrence case. A point at which recurrence occurredis indicated with an arrow. The results are shown in the lower portionof FIG. 34. The horizontal axis represents elapsed months with the dateof operation designated as “0.” Also, the vertical axis represents S/Nratios with the serum from a healthy subject designated as a negativecontrol (N). In the case of patient A, the measured value fluctuatednear S/N=2.5 even after operation and increased on and after therecurrence point. In the case of patient B, the preoperative measuredvalue was very high, but the value decreased after operation andremained unchanged near S/N=2.5. On the other hand, in the case ofpatient E, the lowest measured value, S/N=2.0, was confirmed before andafter operation, and the numerical value gradually decreased. It wasfound based on the above results that while patients with measuredvalues that had remained unchanged near S/N>2.5 after operation have ahigh recurrence risk, and patients with measured values that hadincreased after recurrence, but rapidly decreased after operation tolevels significantly lower than S/N=2.0 may likely have no recurrence.As described above, WFA-binding M2BP is useful as a marker fordiscriminating a high risk group for liver cancer and the anti-M2BPantibody overlay-WFA well plate could be developed as a convenientmeasurement system for detection of the same.

Example 9 Verification of the Effects of Heat Treatment for SpecimensUpon Detection of WFA-Binding M2BP by an ELISA Method

The effects of the presence or the absence of heat treatment forspecimens on the sensitivity of detection of WFA-binding M2BP by anELISA method were confirmed.

A human liver cancer-derived cell line (HepG2 culture supernatant) wasdiluted 10-fold with a 0.2% SDS-containing PBS buffer, followed by 10minutes of heat treatment at 95° C.

M2BP binding to WFA was measured by a method similar to that in 2 ofExample 8 except that an HepG2 culture supernatant (untreated) and anHepG2 culture supernatant (heat-treated) were used as samples so thatthe amount of protein per well was as listed in Table 10. Absorbancemeasured using a plate reader is shown in Table 10 and FIG. 35.

TABLE 10 ng total protein/well Untreated Heat-treated 5000 2.073 0.3653000 1.861 0.275 1000 1.372 0.143 500 1.102 0.097 100 0.398 0.032 500.181 0.03 10 0.047 0.05

It was revealed based on the results in Table 10 and FIG. 35 that theuse of serum that had not been heat-treated as a measurement sample inmeasurement of WFA-binding M2BP by the ELISA method improved measurementsensitivity.

Example 10 Measurement of M2BP by the 2^(nd) Rapid Measurement Method 1.Preparation of Reagents

Preparation of R1 reagent: A solution (buffer C) containing 10 mM HEPES(pH 7.5), 150 mM NaCl, 0.01 mM MnCl₂, 0.1 mM CaCl₂, and 0.08 w/v % NaN₃was prepared and then designated as an R1 reagent.

Preparation of R2 reagent: Magnetic particles (number average particlesize: 2 μm) (hereinafter, referred to as streptavidin-sensitizedmagnetic particles) to which commercially available streptavidin hadbeen immobilized were added to buffer C to a concentration of 0.5 w/v %,and then a biotinylated lectin solution (WFA) was added to the solution.The mixed solution was stirred at room temperature for 30 minutes. Afterstirring, magnetic particles were collected with magnet forprecipitation, and then solution components were discarded. Afterwashing 3 times with buffer C, buffer D (the solution containing 10 mMHEPES (pH7.5), 150 mM NaCl, 0.01 mM MnCl₂, 0.1 mM CaCl₂, 0.1% W/V BSA,and 0.08 w/v % NaN₃) was added so that the concentration of magneticparticles was 0.5 w/v %.

Preparation of R3 reagent: A solution containing a 0.1 U/mL recombinantALP-labeled mouse anti-M2BP monoclonal antibody, 0.1 M MES(2-(N-Morpholino) ethanesulfonic acid, pH6.5), 0.15M NaCl, 1 mM MgCl₂,0.1 mM ZnCl₂, and 0.1 w/v % NaN₃ was prepared and then designated as anR3 reagent.

As an R4 reagent, an R5 reagent, and a washing reagent, the R4 reagent,the R5 reagent, and the washing reagent used in the 2nd rapidmeasurement method in Example 5 were used.

2. Confirmation of Dilution Linearity of a Measurement System Using WFA

A human liver cancer-derived cell line culture supernatant (HepG2culture supernatant) (100 μg/ml) was diluted 10-fold, 100-fold,1000-fold, and 10000-fold with a buffer (PBS), so that diluted sampleswere prepared. Also, 5 μg/ml recombinant human galectin-3BP/MAC-2BP (R&DSYSTEMS) was diluted 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, and64-fold with a buffer (PBS), so that diluted samples were prepared.

Conditions for the operation of a full-automatic immunoassay systemHISCL2000i (Sysmex) were changed to the following conditions.Chemiluminescence intensity (photo count value) was measured for eachdiluted sample.

The R1 reagent (30 μL) and 10 μL of each diluted sample were dispensedto a vessel. After 2.25 minutes of incubation at 42° C., 30 μL of the R2reagent was dispensed, followed by 1.5 minutes of reaction at 42° C.Magnetic particles were collected by magnetic separation, so that thesolution was vacuumed and discarded. The washing reagent was dispensed,magnetic particles were dispersed in and washed with the washing reagentand then collected by magnetic separation, so that the solution wasvacuumed and discarded. This treatment was repeated 3 times. The R3reagent (100 μL) was dispensed, followed by 2.75 minutes of reaction at42° C. Magnetic particles were collected by magnetic separation and thusthe solution was vacuumed and discarded. The washing reagent wasdispensed, magnetic particles were dispersed in and washed with thewashing reagent and then collected by magnetic separation, and thus thesolution was vacuumed and discarded. This treatment was repeated 3times. The R4 reagent (50 μL) was dispensed, 100 μL of the R5 reagentwas dispensed, and then the chemiluminescence intensity was measured.Measurement results for the supernatant of cultured HepG2 cells areshown in Table 11 and FIG. 36 and the measurement results of recombinanthuman galectin-3BP/MAC-2BP are shown in Table 12 and FIG. 37. As shownin these measurement results, WFA measurement system exhibited gooddilution linearity.

TABLE 11 μg/test Luminescence S/N (with respect (10 μL) intensity(count) to diluent) Diluent (PBS) 2,987 1.00 0.0001 3,043 1.02 0.0014,850 1.62 0.01 31,671 10.60 0.1 332,706 111.38

TABLE 12 μg/test Luminescence S/N (with respect (10 μL) intensity(count) to diluent) Diluent (PBS) 2,987 1.00  0.3906 14,456 4.84  0.781326,640 8.92  1.5625 55,721 18.65  3.125 123,939 41.49  6.25 313,747105.04 12.5 663,628 222.17 25 1,822,168 610.03

3. Measurement for Clinical Specimens

Sera from a healthy subject, an HBV-positive hepatic cell carcinomapatient, and an HCV-positive hepatic cell carcinoma patient weremeasured using a full-automatic immunoassay system HISCL2000i underconditions similar to those in “2. Confirmation of dilution linearity ofa measurement system using WFA.” The time required for measurement ofeach specimen was 17 minutes. The results are shown in Table 13.

TABLE 13 S/N Luminescence (with respect to serum intensity (count) fromhealthy subject) Serum from healthy subject 654,663 1 Serum fromHBV-positive 11,317,040 17.3 liver cancer patient Serum fromHCV-positive 17,124,911 26.2 liver cancer patient

It could be confirmed based on the results in Table 13 that M2BP bindingto WFA can be rapidly and precisely measured by the 2^(nd) rapidmeasurement method.

The present invention can be used for producing apparatuses,instruments, or kits for determining liver disease or clinicalconditions of liver disease, distinguishing among clinical conditions ofliver disease, or detecting cirrhosis, for example.

1-30. (canceled)
 31. A reagent for measuring a glycan structure changedMac-2-binding protein in a Mac-2-binding protein (M2BP) contained in ablood sample collected from a subject, comprising: a Wisteria floribundalectin (WFA); and an anti-M2BP antibody.
 32. The reagent according toclaim 31, wherein the WFA selectively binds to the glycan structurechanged Mac-2-binding protein in the blood sample and the anti-M2BPantibody binds to the glycan structure changed Mac-2-binding proteinbound to the WFA.
 33. The reagent according to claim 31, wherein the WFAis immobilized to a particle.
 34. The reagent according to claim 33,wherein the particle is a magnetic particle.
 35. The reagent accordingto claim 31, wherein the WFA is immobilized to a plate substrate. 36.The reagent according to claim 33, wherein the WFA is a biotinylatedWFA, and the particle is a streptavidin or an avidin immobilizedparticle.
 37. The reagent according to claim 31, wherein the anti-M2BPantibody is a labeled anti-M2BP antibody.
 38. The reagent according toclaim 37, wherein a label of the labeled anti-M2BP antibody is alkalinephosphatase.
 39. The reagent according to claim 37, wherein a label ofthe labeled anti-M2BP antibody is deglycosylated alkaline phosphatase.40. The reagent according to claim 31, wherein the anti-M2BP antibody isa deglycosylated anti-M2BP antibody.
 41. The reagent according to claim31, further comprising a Datura stramonium lectin (DSA).
 42. A reagentfor measuring a glycan structure changed Mac-2-binding protein in aMac-2-binding protein (M2BP) contained in a blood sample collected froma subject, comprising: a Wisteria floribunda lectin (WFA) is immobilizeda magnetic particle; and an anti-M2BP antibody labeled by alkalinephosphatase.
 43. A method for measuring a glycan structure changedMac-2-binding protein (M2BP) contained in a blood sample collected froma subject, comprising: providing a reagent comprising a Wisteriafloribunda lectin (WFA) and an anti-M2BP antibody; contacting the bloodsample with the WFA; binding the anti-M2BP antibody to the glycanstructure changed M2BP bound to the WFA; and measuring an amount of theanti-M2BP antibody bound to the glycan structure changed M2BP.
 44. Themethod according to claim 43, wherein the WFA is immobilized to aparticle.
 45. The method according to claim 44, wherein the particle isa magnetic particle.
 46. The method according to claim 43, wherein theWFA is immobilized to a plate substrate.
 47. The method according toclaim 44, wherein the WFA is a biotinylated WFA, and the particle is astreptavidin or an avidin immobilized particle.
 48. The method accordingto claim 43, wherein the anti-M2BP antibody is a labeled anti-M2BPantibody, and the measuring is performed by measuring an amount of alabel of the labeled anti-M2BP antibody bound to the glycan structurechanged M2BP.
 49. The method according to claim 48, wherein the label ofthe labeled anti-M2BP antibody is alkaline phosphatase.
 50. The methodaccording to claim 48, wherein the label of the labeled anti-M2BPantibody is deglycosylated alkaline phosphatase, and the anti-M2BPantibody is a deglycosylated anti-M2BP antibody.